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     

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.     

Accumulation of copper by roots,hypocotyls, cotyledons and leaves of sunflower (Helianthus annuus L.)

The effects of different concentrations of copper sulfate on the growth of and the accumulation of Cu2+ by root, hypocotyl, cotyledon and leaf growth of sunflower (Helianthus annuus L.) were examined in this study. The concentrations of copper sulfate (CuSO4 x 5H2O) used were in the range from 10(-5) to 10(-3) M. Seedlings exposed to 10(-5) M Cu2+ solution exhibited a 33% increase in growth (P < 0.005) when compared with the root length of the control. The seedlings treated with 10(-3) M Cu2+ were significantly inhibited in shoot growth (P < 0.005). The Cu2+ content in roots, hypocotyls, cotyledons and leaves increased with increasing solution Cu2+ concentration. The roots of plants exposed to 10(-3) M Cu2+ accumulated a large amount of Cu (1070 microgram/g DW), and the Cu2+ level was approximately 25 fold higher than that of control. The Cu2+ contents in sunflower roots treated with 10(-4) and 10(-5) M Cu2+ were about 3.3 and 2.6 fold higher than the control, respectively. Also, the Cu2- level of the roots exposed to 10(-3) M Cu2+ was approximately 7.7 and 9.8 fold respectively, in comparison with the roots of plants grown in 10(-4) and 10(-5) M Cu2+. At 10(-3) M Cu2+, the Cu accumulated mainly in the roots (about 73%), and small amounts of Cu2+ (27%) were translocated to the hypocotyls, cotyledons and leaves. The Cu2+ concentration in the roots was less than that of the above parts of seedlings in treated groups with 10(-5) - 10(-4) M Cu2+. H. annuus has potential ability to accumulate Cu without being overly sensitive to Cu toxicity.     

Photosynthesis and chlorophyll fluorescence in sunflower (Helianthus annuus L.) leaves as affected by phosphorus nutrition

The effects of phosphate concentration on plant growth and photosyntheticprocesses in primary leaves of young sunflower (Helianthus annuusL.) plants were examined. Plants were grown for 3 weeks on half-strengthHoagland's solution containing 0, 0.1, 0.5, 1.0, and 3.0 molm^–3 orthophosphate (Pi). It was shown that optimal photosynthesisand the highest light utilization capacity were achieved at0.5 mol m^–3 Pi in the growth medium, which was in goodagreement with the maximum content of organic phosphorus inthe leaves. Low phosphate in the medium inhibited plant growthrate. Phosphate deficiency appreciably decreased photosyntheticoxygen evolution by leaves, the efficiency of photosystem two(PSII) photochemistry and quantum efficiency of PSII electrontransport. High oxidation state of PSII primary electron acceptorQA, at 0.1 mol m^–3 Pi, however, indicates that photosyntheticelectron transport through PSII did not limit photosynthesisin Pi-deficient leaves. The results indicate that diminishedphotosynthesis under sub- and supra-optimal Pi was caused mainlyby a reduced efficiency of ribulose 1, 5-bisphosphate (RuBP)regeneration at high light intensities. These results suggestthat, under non-limiting C0₂ and irradiance, photosynthesisof the first pair of leaves could be diminished by both sub-and supra-optimal phosphorus nutrition of sunflower plants. Key words: Helianthus annuus L, phosphate nutrition, photosynthesis, photochemical efficiency     

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.     

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.     

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     

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     

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.     

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.     

Study of the senescence process in primary leaves of sunflower (Helianthus annuus L.) plants under two different light intensities

Different parameters that vary during leaf development may be affected by light intensity. To study the influence of different light intensities on primary leaf senescence, sunflower (Helianthus annuus L.) plants were grown for 50 days under two photon flux density (PFD) conditions, namely high irradiance (HI) at 350 μmol(photon) m^?2 s^?1 and low irradiance (LI) at 125 μmol(photon) m^?2 s^?1. Plants grown under HI exhibited greater specific leaf mass referred to dry mass, leaf area and soluble protein at the beginning of the leaf development. This might have resulted from the increased CO₂ fixation rate observed in HI plants, during early development of primary leaves. Chlorophyll a and b contents in HI plants were lower than in LI plants in young leaves. By contrast, the carotenoid content was significantly higher in HI plants. Glucose concentration increased with the leaf age in both treatments (HI and LI), while the starch content decreased sharply in HI plants, but only slightly in LI plants. Glucose contents were higher in HI plants than in LI plants; the differences were statistically significant (p<0.05) mainly at the beginning of the leaf senescence. On the other hand, starch contents were higher in HI plants than in LI plants, throughout the whole leaf development period. Nitrate reductase (NR) activity decreased with leaf ageing in both treatments. However, the NR activation state was higher during early leaf development and decreased more markedly in senescent leaves in plants grown under HI. GS activity also decreased during sunflower leaf ageing under both PFD conditions, but HI plants showed higher GS activities than LI plants. Aminating and deaminating activities of glutamate dehydrogenase (GDH) peaked at 50 days (senescent leaves). GDH deaminating activity increased 5-fold during the leaf development in HI plants, but only 2-fold in LI plants. The plants grown under HI exhibited considerable oxidative stress in vivo during the leaf senescence, as revealed by the substantial H₂O₂ accumulation and the sharply decrease in the antioxidant enzymes, catalase and ascorbate peroxidase, in comparison with LI plants. Probably, systemic signals triggered by a high PFD caused early senescence and diminished oxidative protection in primary leaves of sunflower plants as a result.     

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     

Seawater irrigation: antioxidant defence responses in leaves and roots of a sunflower (Helianthus annuus L.) ecotype

Salinity is a widespread environmental stress for crop plants. It is common in arid, semiarid, and coast regions. In those environments, seawater infiltrations can occur or the sea provides the only source of water for irrigation. The effects of 10% and 20% seawater in nutrient solutions were studied in 30 day-old plants of sunflower (Helianthus annuus L.) ecotype Katharina Piacenza. Growth parameters, ascorbate and glutathione contents, and the activities of ascorbate peroxidase and glutathione reductase were determined in shoots and roots. The results showed antioxidative responses of the ecotype to both salt treatments. The different activity patterns of antioxidant molecules and enzymes in the leaves and roots suggested a different kind of reaction to the two seawater concentrations.     

Effect of field limestone applications on cadmium content of sunflower (Helianthus annuus L.) leaves and kernels

Nonoilseed sunflower (Helianthus annuus L.) is naturally higher in cadmium (Cd) than many other grain crops. Because raising soil pH usually depresses Cd uptake by most species, a study was designed to determine if application of agricultural limestone to neutralize soil acidity would decrease Cd uptake by sunflower plants grown on different soils in the production area of North Dakota. The field experiments were conducted at 3 locations in 1991 and 2 locations in 1992. At each site, limestone was applied to bring soil pH to 6.5–7.0, or an additional 45 Mg ha^-1 more limestone was applied, and these two treatments were compared to no-lime control. Commercial nonoilseed hybrid 954 was planted in these experiments. The rapid short-term lime-soil reaction occurred in first 12 weeks following limestone application. Mean kernel Cd concentration for each treatment varied from 0.35 to 1.45 mg kg^-1 DW in the first year of the experiments, and from 0.37 to 1.23 mg kg^-1 DW in the experiments of 1992 across all locations. Large variations in kernel Cd levels between locations were obtained. There were no significant differences among control and limestone treatments for kernel Cd, seedling leaf Cd and diagnostic leaf Cd within each location, respectively. In regression analysis, we found that kernel Cd level correlated with diagnostic leaf Cd concentration in each treatment, but poor correlations were obtained among other variables. These results indicated that limestone application did not reduce Cd uptake and transfer to kernels of sunflower, in contrast with most species studied.     

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.     

Investigation of the limitations to photosynthesis induced by leaf water deficit in field-grown sunflower (Helianthus annuus L.)

Abstract. The diurnal cycling of leaf water potential (Ψ_leaf) in field-grown sunflower ( Helianthus annuus ) was used to investigate the cause of water deficitinduced limitation of net photosynthesis. Daily midafternoon decreases in Ψ_leaf of up to 1.5 MPa and in net photosynthesis of up to 50% were typical for irrigated sunflower during seed filling. These midafternoon values were lowered an additional 0.6 to 0.8 MPa by prolonged drought treatment. There was a nearly linear relationship between the decline in net photosynthesis and reductions in leaf conductance over the course of the day. Thus, it was unexpected to find that the low, midafternoon rates of photosynthesis were associated with the highest intercellular CO₂ concentrations. These and other observations suggest that the daily decline in photosynthesis represents a 'down regulation' of the biochemical demand for CO₂ that is coordinated with the diurnally developing need to conserve water, thus establishing a balanced limitation of photosynthesis involving both stomatal and non-stomatal factors. There were no indications that either short term (i.e. diurnal declines in Ψ_leaf) or long term (i.e. drought treatment) water deficits caused any damage or malfunctioning of photosynthesis. Rather, both the daily declines in photosynthesis and the nearly 25% decrease in leaf area induced by prolonged drought appeared to be well-controlled adaptive responses by field-grown sunflower plants to limited water availability.     

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.     

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