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.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

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.     

Agrobacterium-mediated transformation of sunflower (Helianthus annuus L.) shoot apices: transformation patterns

Apical segments of embryonic axes of sunflower (Helianthus annuus L.) embryos were submitted to co-culture experiments with a disarmed strain of Agrobacterium tumefaciens, harbouring a plasmid coding for the marker enzyme -glucuronidase. The expression patterns of this marker were analysed at different developmental stages of the regenerated shoots. The results are consistent with the hypothesis that transformed shoots originate from transformation events that have occurred within the existing meristems. Two of the resulting chimaeric plants have been analysed in detail, and some representative gene integration patterns are presented.     

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     

Relationships between root permeability to water,leaf conductance and transpiration rate in sunflower (Helianthus annuus L.) cultivars

Summary The relations between leaf conductance (g_l) transpiration rate and root permeability to water (R_p) of three sunflower (Helianthus annuus L.) cultivars grown in a controlled environment cabinet are described.No differences in transpiration rates were found but it was shown that plants with low values of R_p have active stomatal closure with favourable consequences for water use efficiency under water limiting conditions.R_p was estimated by applying hydrostatic pressure on the root system. Values of R_p per unit root volume ranged from 0.34×10^–5 to 16.75×10^–5 (s MPa^–1). There were significant inter-cultivar differences (P<0.05) in R_p and g_l and an inverse correlation between R_p and the maximum values cf g_l within cultivars.Pressure applied on the root system is proposed as a useful tool for the determination of differences in the root permeability to water amongst sunflower cultivars.     

Changes in mass and dimensions of sunflower (Helianthus annuus L.) Achenes and seeds due to carbonization

When analyzing sunflower (Helianthus annuus L.) remains, which are often carbonized, archaeobotanists commonly differentiate between wild and domesticated achenes and seeds based on the measured length (L) and width (W) or the calculated index L*W. Carbonization reduces the dimensions. To compensate for these reductions, archaeobotanists use a single correction factor proposed by Richard Yarnell (1978) for all cases. The use of a single correction factor can bias the reconstructed dimensions as carbonization is a highly variable process. The current study determines the relationship between carbonization and the dimensions of length and width. Measurements established that a decrease of 2.5-22.5% in achene length and 10-29% in achene width can occur, depending on temperature, heating rate, and variety. For seeds, temperature is of most importance, and shrinkage ranges from 0-27% for the length and from 0-20% for the width. These ranges make the use of a single correction factor problematic. A method is developed in which reflectance (an optical property applied in coal technology to determine coal rank) is used to measure the carbonization temperature, and in turn the shrinkage can be calculated. Subsequently, correction factors are calculated to reconstruct the original length and width. When applied to an assemblage of carbonized sunflower achenes, the newly developed method shows that the Yarnell single correction factor may bias the dimensions towards classifications of “wild” or “ruderal” forms of sunflower     

Effects of foliar applied gibberellic acid and benzyladenine upon yield components in sunflower (Helianthus annuus L.)

Sunflower yield is determined by seed number/m^–2 and by achene weight. Frequently, a high percentage of empty achenes in the inner portion of the capitulum, probably due to a reduced vascularization of that section of the flower head, decreases final yield. The objective of the present research is to determine if foliarly applied gibberellic acid (GA) and benzyladenine (BA) can enhance the vascularization in the inner portion of the capitulum, improving photoassimilate translocation. Field experiments were conducted during 1989/90 with hybrid SPS 894 and during 1990/91 with hybrid ACA 882. GA (150 mg/l^–1), BA (150 and 250 mg/l^–1) and GA 150+BA 150 mg/l^–1 each were foliarly applied 20, 40, or 60 days after emergence. For both seasons and hybrids plant growth regulator (PGRs) applications significantly reduced the percentage of empty achenes, increased achene weight, achene weight (× 1000) and achene number in the inner portion of the capitulum and in the middle and outer portion during 1990/91. A 25% increase in seed yield was achieved due to PGR application and the capitulum partition index (achene weight/receptacle weight^–1, CPI) was significantly increased due to an improvement in photoassimilate distribution. A distribution model was derived showing that preferential allocation of photoassimilates in the outer portion of the capitulum can be modified by PGR application, demonstrating that photoassimilate distribution is under hormonal control.Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET)     

Leaf responses to soil water deficits: Comparative sensitivity of leaf expansion rate and leaf conductance in field-grown sunflower (Helianthus annuus L.)

The relative importance of changes in leaf expansion rate (LER) and leaf conductance (g₁) in the control of crop transpiration depends primarily on their sensitivity to soil water deficits. The aim of this paper was to quantify the responses of LER and g₁ to soil water deficits in sunflower (Helianthus annuus L.) under conditions of moderate (spring) and high (summer) evaporative demand. Soil water content, g₁, and LER were measured in dryland (DRY) and daily-irrigated (WET) crops established on a deep sandy-loam (Typic Xerofluvent) in a Mediterranean environment. There was no difference between g₁ of DRY and WET plants (p>0.20) in contrast with a highly significant difference in LER (p<0.001). Even under the harsh conditions of the summer experiment, g₁ did not respond to water deficit in a ten-day period in which LER of DRY plants was reduced to approx. 30% of that measured in WET controls. This field study indicates that g₁ plays at most a minor role in the control of sunflower transpiration in the pre-anthesis period and confirms the importance of leaf expansion in the regulation of gas exchange of expanding canopies subjected to soil water deficits.     

Structural analysis of colonies derived from sunflower (Helianthus annuus L.) protoplasts cultured in liquid and semi-solid media

Summary Colonies have been generated from cotyledon protoplasts of sunflower (Helianthus annuus L.) and transferred to solid regeneration medium. The development of these protoplasts was strikingly dependent on their physical environment (liquid or solidified medium) and the nature of the gelling agent (agarose, alginate). In particular, extremely compact multicellular structures developed in agarose solidified medium, similar to those observed in cultures of hypocotyl- and petiole-derived sunflower protoplasts (termed proembryoids). A detailed study at the histological level, however, indicates that these structures are degenerative, rather than embryonic in nature. The quality of the colonies, with regard to their regeneration potential, appears to be best in alginate solidified medium.Abbreviations MS medium of Murashige and Skoog (1962) - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine     

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.     

Light-associated nitrogen distribution profile in flowering canopies of sunflower (Helianthus annuus L.) altered during grain growth

In vegetative canopies of many species, the vertical gradient of lamina nitrogen concentration (NW) parallels the profile of light distribution in such a way that the actual nitrogen partitioning approaches the optimum pattern for canopy photosynthesis. This paper evaluates the hypothesis that a strong sink for nitrogen, viz. growing grain, affects the pattern of lamina nitrogen distribution usually described for vegetative canopies. The light and NW profiles of sunflower (Helianthus annuus L.) crops were characterised from anthesis to physiological maturity. The factorial combination of two plant populations (2.4 and 4.8 plants m^–2) and two levels of nitrogen supply (0 and 5 g N m^–2) were the sources of variation for NW and light profiles. Before the onset of nitrogen accumulation in grain, the pattern of NW was similar to that described for other species and it was related to the distribution of light in the canopy. Important changes in the profile of NW occurred during grain filling that were unrelated to the light regime. Nitrogen was mobilised from leaves in all positions in the canopy and the rate of NW change was greater in leaves closer to the grain, which were also the leaves where nitrogen was more concentrated. It is concluded that the physiological mechanisms involved in determining the distribution of leaf nitrogen in vegetative canopies do not apply to sunflower during grain filling.     

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.     

Leaf expansion of four sunflower (Helianthus annuus L.) cultivars in relation to water deficits. II. Diurnal patterns during stress and recovery

Abstract. Leaf expansion of four sunflower cultivars ( Helianthus annuus L. cvs. Hysun 31, Havasupai, Hopi and Seneca) was monitored continuously in a growth cabinet through the final stages of a drying cycle and then throughout the first 2 days after rewatering in order to study the responses of leaf expansion to water deficits. Comparable plants were also measured throughout a diurnal cycle in a glasshouse.
In the cabinet, leaf extension was faster in the dark than in the light, but an extended dark period suppressed leaf extension. At similar leaf water potentials, the rate of leaf extension was greater in the light than in the dark, but as the osmotic potential was lower in the light than in the dark, the relationship between turgor pressure and leaf extension rate was similar in both environments. Throughout the drying and recovery cycles turgor and leaf extension rate was positively correlated: no significant differences among cultivars were observed.
In the plants grown and measured in the glasshouse, leaf expansion occurred at lower leaf water potentials in stressed than in unstressed plants, but the relationship between leaf expansion and turgor was similar in both stressed and unstressed plants as a result of a lowering of the osmotic potential in the former. Diurnal turgor maintenance resulting from osmotic adjustment was almost half that occurring during a complete drying cycle. During the day, the leaf expansion rate increased linearly with turgor pressure in all cultivars: the expansion rate per unit turgor pressure was greater in the glasshouse than in the growth cabinet. Nocturnal leaf expansion in the stressed and unstressed plants was not, however, correlated with turgor pressure.     

Bacterial antagonists of Sunflower (Helianthus annuus L.) fungal pathogens

Bacteria isolated on nutrient agar and King's medium B from sunflower leaves, crown and roots inhibited in vitro growth of the leaf spot and wilt pathogens Alternaria helianthi, and Sclerotium rolfsii, respectively, and also the root rot pathogensRhizoctonia solani and Macrophomina phaseolina. Antagonistic bacteria from leaves were mainly actinomycetes and pigmented Gram-positive bacteria, while those from roots and crowns were identified asPseudomonas fluorescens-putida, P. maltophilia, P. cepacia, Flavobacterium odoratum andBacillus sp. In soil bioassays, when used as seed inoculum in the presence ofS. rolfsii, P. cepacia strain N24 increased significantly the percentage of seedling emergence. Bacterial strains which exhibited broad spectrum in vitro antagonistic activity were tested for colonisation of sunflower roots, when used as a seed inoculum. Good colonisers (10⁴ to 10⁶ bacteria/g root) were consistent in their ability to reduce disease and fungal wilt. A seedling having a primary root length < 5 cm with fewer lateral roots, necrosed cotyledons or crown and a wilted shoot indicated its diseased status. On an average, only 30% of seedlings were diseased when treated with the antagonistic strains, in the presence of the pathogen, while 60% of the seedlings were diseased in the presence of the pathogen alone. In microplots treated with strain N24, only 1 to 3% of the seedlings were wilted, while 14% of the seedlings were wilted in the presence of the pathogen alone. The results obtained show that bacterial antagonists of sclerotial fungi can be used as seed inocula to improve plant growth through disease suppression     

Effects of leaf position and nitrogen supply on the expansion of leaves of field grown sunflower (Helianthus annuus L.)

Leaf growth responses to N supply and leaf position were studied using widely-spaced sunflower plants growing under field conditions. Both N supply (range 0.25 to 11.25 g added N per plant) and leaf position significantly (p=0.001) affected maximum leaf area (LA_max) of target leaves through variations in leaf expansion rate (LER); effects on duration of expansion were small. Specific leaf nitrogen (SLN, g N m^-2) fell quite rapidly during the initial leaf expansion phase (LA < 35% LA_max) but leveled off during the final 65% increase of leaf area. This pattern held across leaf positions and N supply levels. Leaf nitrogen accumulation after 35% LA_max continued up to achievement of LA_max; reductions in the higher SLN characteristic of the initial phase were insufficient to cover the nitrogen requirements for expansion during the final phase. LER in the quasi-linear expansion phase (35 to 100% of LA_max) was strongly associated with SLN above a threshold that varied with leaf position (mean 1.79±0.225 g N m^-2). This contrasts with the response of photosynthesis at high irradiance to SLN, which has previously been shown to have a threshold of 0.3 g N m^-2; in the present work saturation of photosynthetic rate was evident when SLN reached 1.97 g N m^-2. Thus, once the area of a leaf exceeds 35% of LA_max, expansion proceeds provided SLN values are close to the levels required for maximum photosynthesis. However, growth of leaves during the initial expansion phase ensures a minimum production of leaf area even at low N supply levels.     

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.     

Tissue culture and plant regeneration from sunflower (Helianthus annuus) and interspecific hybrids (H. tuberosus x H. annuus)

A method is described for the culture and regeneration of plants from callus of sunflower (Helianthus annuus) andH. annuus x H. tuberosus hybrids. Immature embryos proved to be the only explant which consistently gave regenerable cultures in all genotypes. The most responsive embryos were approximately 12 mm² in area. Genotype had a significant effect on the capacity of cultures to regenerate. Some regeneration was also obtained from cultures of tuber tissue but only from one genotype,H. tuberosus x H. annuus cross 200. None of theH. annuus accessions gave regenerable callus from root tissue. Difficulties included the premature initiation of flowering of regenerating shoots and the frequent occurence of "vitreous" plantlets which could not be transplanted successfully to soil. Some amelioration of both these problems was achieved by replacing inorganic nitrogen partially with amino acids. More effective reduction of these difficulties was accomplished by the addition of 10, 30 and 100 M phloridzin, esculin or naringin.Abbreviations BAP 6-benzylaminopurine; zeatin, trans-6-(4-hydroxy-3-methyl-but-2-enyl) aminopurine; kinetin, 6-furfurylaminopurine - IAA indole-acetic acid - NAA naphthyl acetic acid     

Exposure to low levels of photosynthetically active radiation induces rapid increases in palisade cell chloroplast volume and thylakoid surface area in sunflower (Helianthus annuus L.)

Summary Sudden changes in photoactive radiation (PAR) (wavelength, 400–700 nm) induces rapid surface area changes in chloroplast thylakoid membranes. Although this response may have important photo-acclimative functions for the plant, little is known about the mechanisms by which changes in irradiance are detected or how thylakoid membranes actually increase or decrease surface area. Knowledge of the time required for significant changes in thylakoid area would help eliminate or support several possible mechanisms that may be involved in this aspect of photo-acclimation in plants. Leaf tissues were acclimated to a PAR of 500 mol quanta per m² per s then exposed to low irradiance (PAR, 50 mol quanta per m² per s) and sampled at 5, 15, 30, and 60 min post exposure. Tissue and cell structure were quantified and results showed a significant increase in the surface-to-volume ratio and surface area per unit of standard leaf volume for both appressed and nonappressed thylakoids within 5 min of exposure to low irradiance. On the basis of the ratios of appressed to nonappressed thylakoids, the surface area of the nonappressed thylakoids was found to increase faster than that of the appressed thylakoids throughout the sample period. The portion of the appressed thylakoids in contact with the stroma was defined as margin thylakoids. Margin thylakoid surface-to-volume ratio did not change relative to the high-irradiance control during the sample period but did remain significantly lower than the low-irradiance control during the sample period. The ratio of appressed to margin thylakoids indicated a broadening and shortening of the appressed thylakoid stack within the first 5 min of low-irradiance exposure. The rapidity of the shade response indicates that the early events in this response probably do not directly involve gene activation pathways.Abbreviations PAR photosynthetically active radiation - Sv surface to volume density - Vv volume density - UV-B ultraviolet B radiation     

Molecular marker analysis of Helianthus annuus L. 2. Construction of an RFLP linkage map for cultivated sunflower

A detailed linkage map of Helianthus annuus was constructed based on segregation at 234 RFLP loci, detected by 213 probes, in an F₂ population of 289 individuals (derived from a cross between the inbred lines HA89 and ZENB8). The genetic markers covered 1380 centiMorgans (cM) of the sunflower genome and were aranged in 17 linkage groups, corresponding to the haploid number of chromosomes in this species. One locus was found to be unlinked. Although the average interval size was 5.9 cM, there were a number of regions larger than 20 cM that were devoid of markers. Genotypic classes at 23 loci deviated significantly from the expected ratios (121 or 31), all showing a reduction in the ZENB8 homozygous class. The majority of these loci were found to map to four regions on linkage groups G, L and P.