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.     

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.     

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     

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.     

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.     

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     

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     

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     

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.     

Photosynthetic characteristics of mesophyll cells isolated from cladophylls ofAsparagus officinalis L.

Intact mesophyll cells can be rapidly isolated from the cladophylls ofAsparagus officinalis by gentle scraping with a plastic card, the yield being higher than 80% on a chlorophyll basis. The cells can be stored for at least 24h without loss of photosynthetic capacity and were found to be stable under a variety of conditions. In contrast to cell preparations from other plant species, photosynthetic activity was little affected by the presence of sorbitol as an osmoticum up to a concentration of 1.5 M. Similarly, the pH value of the medium influenced photosynthesis to only a small extent at a constant [CO₂] of 200 M. The response of the cells' photosynthetic capacity to light, temperature and CO₂ concentration was similar to those reported for isolated cells from other plant species. Isolated cells ofA. officinalis can be used under a large range of conditions which gives them a measure of flexibility not possible with most plant cells which have sharply defined optimal conditions for photosynthesis. The isolated cells have a photosynthetic capacity of 40–60% of that of the intact cladophyll. The loss of photosynthetic activity observed upon isolation could not be accounted for by breakage of the cells. Virtually all of the cells were shown to be intact on the basis of Evans Blue exclusion and more than 80% of the cells contained intact chloroplasts and vacuoles. The entire loss of photosynthetic activity could be accounted for by a decrease in sucrose synthesis rather than by an equal decrease in the synthesis in all products. A six- to seven fold increase in the level of¹⁴C in hexose phosphates in the isolated cells supports the notion of inhibition of the sucrose-synthesis pathway.     

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)     

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.     

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.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

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.     

The intercellular compartmentation of metabolites in leaves of Zea mays L.

Sap extracted from attached leaves of two-to three-week-old maize plants witt the aid of a roller device was almost devoid of bundle-sheath contamination as judged by the distribution of mesophyll and bundle-sheath markers. The extraction could be done very rapidly (less than 1 s) and the extract immediately quenched in HClO₄ or reserved for enzyme assay. Comparison of the contents of metabolites in intact leaves and in the leaf extract allowed estimation of the distribution of metabolites between the bundle-sheath and the mesophyll compartments. Substantial amounts of metabolites such as malate and amino acids were present in the non-photosynthetic cells of the midrib. In the illuminated leaf, triose phosphate was predominantly located outside the bundle-sheath while the major part of the 3-phosphoglycerate was in the bundle sheath. The results indicate the existence of concentration gradients of triose phosphate and 3-phosphoglycerate in the leaf which are capable of maintaining carbon flow between the mesophyll and bundle-sheath cells during photosynthesis. There was no evidence for the existence of a gradient of pyruvate between the bundle-sheath and the mesophyll cells.     

Photosynthetic characteristics of C4 trait in chlorina mutant of rice (Oryza sativa L.)

Biao 810S is a chlorina mutant of the thermosensitive genic male sterile (TGMS) rice. We compared photosynthetic characteristics of these two lines. The contents of chlorophylls and carotenoids in Biao 810S were approximately half of those in 810S. However, the net photosynthetic rate (P _N) of Biao 810S was higher than that of 810S under high irradiance or low concentration of carbon dioxide, and the photon quantum efficiency was higher than that of 810S. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase in Biao 810S was only 69.80 % of that in 810S, but the activities of phosphoenolpyruvate carboxylase and NADP-malic enzyme were 79.50 and 69.06 % higher than those of 810S, respectively, suggesting that the efficiency of photon energy utilization in Biao 810S was enhanced by reduction of thermal dissipation and increase of electron transfer rate to generate sufficient assimilation power for the dark reactions. Consequently, the increased activities of C₄ photosynthetic enzymes lead to more effective fixation of CO₂ and the synergistic effect of light and dark reactions contributed to the higher P _N of Biao 810S.     

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     

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.