Effects of potassium and gibberellic acid on stem growth of whole sunflower plants

Abstract The effects of gibberellic acid (GA₃) on whole sunflower (Helianthus annuus L.) plants grown at three potassium (K) levels (0.0, 0.5 and 5.0 mM) were studied. A tenfold increase in the length of the first internode was observed when plants grown without K were treated with GA₃. The uneven K distribution along the plant (higher K content in the higher internodes) was enhanced by GA₃ treatment. Gibberellic acid increased the content of reducing sugars, especially in K-deficient plants. An increase in the K level in the nutrient solution resulted in a decrease of the osmotic potential of stem segments. Osmotic potential differences within the elongating first internode were increased by GA₃ treatment.     

Effects of nitrate on influx, efflux and translocation of potassium in young sunflower plants

Influx, efflux and translocation of K⁺(⁸⁶Rb) were studied in the roots of sunflower seedlings ( Helianthus annuus L. cv. Uniflorus) treated with 0–4.0 m M NO₃⁻ during a 9 day growth period or a 24 h pretreatment period. Roots treated with high levels of NO₃⁻ absorbed and translocated more K⁺(⁸⁶Rb) than seedlings treated with low levels of NO₃⁻. The content of K⁺ in the shoots was, however, higher in seedlings treated with low levels of NO₃⁻, indicating a low rate of retranslocation of K⁺ in those plants. K⁺(⁸⁶Rb) efflux was highest into the low-NO₃⁻ solutions. All effects on K⁺(⁸⁶Rb)-fluxes were more obvious in high-K plants than in low-K plants. The results are discussed in relation to the Dijkshoorn-Ben Zioni hypothesis for K⁺+ NO₃⁻-uptake and translocation in plants.     

Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi₃, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi₃ gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi₃ mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.     

Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirsutum L.) was transformed by the EHA101 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and β-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue, and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assay. Shoots regenerated from excised shoot meristems or their halves were cultured for 4–6 weeks to obtain rooted plants, which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus, a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 462–467. The text was submitted by the authors in English.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

Sequential response of whole plant water relations to prolonged soil drying and the involvement of xylem sap ABA in the regulation of stomatal behaviour of sunflower plants

Sunflower plants ( Helianihus animus cv. Tall Single Yellow} were grown in the greenhouse in drain pipes (100 mm inside diameter and 1 m long) rilled with John Innes No. 2 compost. When the fifth leaf had emerged, half of the plants were left unwatered for 6 days, rewatered for 2 days and then not watered for another 12 days. Measurements of water relations and abaxial stomatal conductance were made at each leaf position at regular intervals during the experimental period. Estimates were also made of soil water potentials along the soil profile and of ABA concentrations in xylem sap and leaves.
Soil drying led to some reduction in stomatal conductance alter only 3 days but leaf turgors were not reduced until day 13 (6 days after rewatering). When the water relations of leaves did change, older leases became substantially dehydrated while high turgors were recorded in younger leaves. Leaf ABA content measured on the third youngest leaf hardly changed over the first 13 days of the experiment, despite substantial soil drying, while xylem ABA concentrations changed very significantly and dynamically as soil water status varied, even when there was no effect of soil drying on leaf water relations. We argue that the highest ABA concentrations in the xylem, found as a result of substantial soil drying, arise from synthesis in both the roots and the older leaves, and act to delay the development of water deficit in younger leases.
In other experiments ABA solutions were watered on to the root systems of sunflower plants to increase ABA concentrations in xylem sap. The stomatal response to applied ABA was quantitatively very similar to that to ABA generated as a result of soil drying. There was a log-linear relationship between the reduction of leaf conductance and the increase of ABA concentration m xylem sap.     

根癌农杆菌介导的转新城疫病毒融合蛋白基因水稻植株的获得

利用转基因植物作为生物反应器可以表达重组蛋白、生产外源蛋白质,也可以成为动物疫苗的廉价生产系统。以编码新城疫病毒融合蛋白(NDV-F)的基因为外源基因,以玉米泛素蛋白(Ubi)启动子为启动子,以潮霉素磷酸转移酶(HPT)基因作为选择标记基因,β-半乳糖苷酸酶(GUS)基因作为报告基因构建了适宜于农杆菌介导转化水稻的表达质粒pUNDV,并通过农杆菌介导转化水稻,获得了多株转基因植株。通过PCR分析和GUS活性检测,证实含有NDV-F基因的T-DNA已整合到水稻核基因组中,为研制廉价安全的转基因水稻新城疫基因工程疫苗奠定了基础。     

The validity of in vitro screening methods in the search for fungal antagonists of Sclerotinia sclerotiorum causing wilt of sunflower

A multi‐test screening system identified 63 fungal isolates with high in vitro biocontrol activity against Sclerotinia sclerotiorum. A bioassay method was developed, using sunflower seedlings growing in an unsterilized loam mixture. Twenty‐six isolates were tested in a series of five bioassay tests and a significant correlation (P < 0.01) was found between sclerotial infection in vitro and the number of healthy plants in vivo. Conversely, activity in an in vitro mycelial overgrowth test was not significantly correlated with activity in vivo. However, some isolates showing only mycelial activity still exerted significant disease control in both the bioassays at Littlehampton and in three additional bioassays at Sittingbourne. Only one isolate not previously reported showed significant activity in both sets of bioassays and the lack of consistency in disease control activity by all other isolates, and biocontrol agents in general, was deemed a major barrier to their use.     

Glyphosate inhibition of ferric reductase activity in iron deficient sunflower roots

Iron (Fe) deficiency is increasingly being observed in cropping systems with frequent glyphosate applications. A likely reason for this is that glyphosate interferes with root uptake of Fe by inhibiting ferric reductase in roots required for Fe acquisition by dicot and nongrass species. This study investigated the role of drift rates of glyphosate (0.32, 0.95 or 1.89 mm glyphosate corresponding to 1, 3 and 6% of the recommended herbicidal dose, respectively) on ferric reductase activity of sunflower (Helianthus annuus) roots grown under Fe deficiency conditions. Application of 1.89 mm glyphosate resulted in almost 50% inhibition of ferric reductase within 6 h and complete inhibition 24 h after the treatment. Even at lower rates of glyphosate (e.g. 0.32 mm and 0.95 mm), ferric reductase was inhibited. Soluble sugar concentration and the NAD(P)H oxidizing capacity of apical roots were not decreased by the glyphosate applications. To our knowledge, this is the first study reporting the effects of glyphosate on ferric reductase activity. The nature of the inhibitory effect of glyphosate on ferric reductase could not be identified. Impaired ferric reductase could be a major reason for the increasingly observed Fe deficiency in cropping systems associated with widespread glyphosate usage.     

Cloning,sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.     

Function of the ascorbate-glutathione cycle in aged sunflower seeds

The function of the ascorbate-glutathione (AsA/GSH) cycle was analyzed in seeds of sunflower ( Helianthus annuus L. cv. Peredovik) subjected to accelerated ageing at 43°C and 75% relative humidity for 1 to 11 days. The study was performed using dry seeds and seeds hydrated by imbibition in distilled water for 4 h at 25 °C. Lipid peroxidation was also determined by measuring the malondialdehyde (MDA) level. As the ageing period increased, a progressive loss of seed viability became increasingly evident. Even though high levels of MDA were delected, the MDA level did not change during accelerated ageing, suggesting that lipid peroxidation might occur to some extent. The study of the ascorbate/glutathione (AsA/GSH) cycle revealed that the GSH system is the major detoxifying mechanism in both dry and imbibed sunflower seeds. The GSH system is mainly located in the embryo, and its protective role is mediated by reactions that consume the GSH pool and, thereby, minimize the increase of the oxidized form (GSSG). Seed imbibition activates cellular metabolism and allows some antioxidant enzymes like glutathione reductase (EC 1,6,4,2) to act upon toxic agents. These reactions provide a reducing status, so that repair of damage becomes possible. However, prolonged ageing conditions (11 days) result in an irreversible damage, as evidenced by the appearance of dead seeds when the germination period ended. Multiple regression analysis revealed the effectiveness of the GSH system in aged seeds, especially upon imbibition and until the AsA/GSH cycle became completely functional.     

Ageing-induced changes in glutathione system of sunflower seeds

The glutathione system is thought to be involved in defence mechanisms present in plant tissues. The efficacy of this system was evaluated in large seeds of sunflower ( Helianthus annuus L. cv. Peredovik) in response to accelerated ageing (43°C/75% relative humidity from 1 to 11 days). Differences between the embryo axis and cotyledons in relation to the glutathione system were also investigated. Additionally, lipid peroxidation was determined by measuring the malondialdehyde (MDA) content. All assays were performed using dry seeds and seeds subsequently hydrated by imbibition in distilled water for 12 h at 25°C. Accelerated ageing caused a marked decrease in seed viability, accompanied by an increase in mean germination time. There were no changes in total glutathione in dry seeds. However, the distribution in its reduced (GSH) and oxidized (GSSG) forms revealed that ageing produced a slow conversion from GSH to GSSG. As the ageing period increased, this effect was accompanied by a decrease in glutathione reductase (GR, EC 1.6.4.2) activity. The results also indicated that the GSH system exerts a different response in the embryo axis as compared with the cotyledon: (1) the GSH levels decreased less in the cotyledons than in axes of aged seeds, and (2) the GSSG level in cotyledons was independent of ageing, while its amount increased in aged embryo axes. These different responses, in conjunction with the lower MDA levels in large as compared with small seeds, indicate a possible protective role of the reserve lipids. The efficacy of the GSH system in aged seeds was associated with seed viability, as revealed by multiple regression analysis. Upon imbibition, aged seeds were able to restore their GSH levels, reaching values approximating those of unaged seeds.     

Purification and characterisation of an inhibitor of a cathepsin B-like proteinase from sunflower seed

Cathepsin B is a vitally important enzyme in various physiological processes and in tumor invasion and metastasis. A cathepsin B inhibitor, HCB-SunI, was identified and purified from sunflower seeds, Helianthus annuus, using ammonium sulfate precipitation and two steps of conventional chromatography. The molecular mass of HCB-SunI was estimated to be 12 kDa by SDS-PAGE and 12.32 kDa by MALDI TOF MS. Its N-terminal amino acid sequence was determined to be: PYGGGGTESG. HCB-SunI not only inhibited Helicoverpa cathepsin B (HCB) but also decreased the growth of HeLa and glioma cells by 7 ～ 27% and 6 ～ 22%, respectively, when the cells were grown in a final concentration of 0.002 ～ 0.008 μM inhibitor.     

Interaction between sunflower plants and five isolates of Plasmopara halstedii on the level of pathogenicity

The interaction between sunflower plants showing a high level of quantitative resistance and five Plasmopara halstedii (the causal agent of downy mildew) isolates of several races were studied using five single zoosporangium isolates per pathogen isolate. Aggressiveness criteria were analyzed for 25 P. halstedii single zoosporangium isolates. Based on the reaction for the P. halstedii isolates to four sunflower hybrids H1–H4 varying only in their downy mildew resistance genes, there were differences in virulence spectrum in pathogen isolates. Analysis of five single zoosporangium isolates for P. halstedii isolates showed significant variability within pathogen isolate for all aggressiveness criteria but not for all pathogen isolates. The hypothesis explaining the interaction between P. halstedii and its host plant was discussed on the level of pathogenicity.     

Responses of photosynthesis and carbohydrate-partitioning to limitations in nitrogen and water availability in field-grown sunflower*

Abstract. Sunflower plants (Helianthus annuus L., cv. CGL 208) were field-grown in adjacent plots of varying resource availability. Control plants received irrigation (on a 4–5 d interval) and high levels of fertilizer nitrogen. Nutrient-stress (N-stress) plants received control levels of irrigation but no nutrient amendments and were determined to be nitrogen-limited. Water-stress (H₂O-stress) plants received control levels of fertilizer nitrogen, but no irrigation after approximately 6 weeks of plant growth. Both stress treatments reduced maximum and diurnal net photosynthesis (A) but resulted in different physiological or biochemical adjustments that tended to maintain or increase A per unit of resource (nitrogen or water) in shortest supply while decreasing the ratio of A per unit of abundant resource. Nutrient-stress reduced total foliar nitrogen, foliar chlorophyll, and initial and total RuBPCase activities, thereby enhancing or preserving photosynthetic nitrogen-use efficiency (NUE), defined as the maximum A observed per unit of leaf nitrogen, relative to the control and H₂O-stress treatments. In addition, N-stress reduced photosynthetic water-use efficiency (WUE), defined as the ratio of A to stomatal conductance to water vapour (g). The slope of A versus g increased with H₂O-stress. In addition, sunflower plants responded to H₂O-stress by accumulating foliar glucose and sucrose and by exhibiting diurnal leaf wilting, which presumably provided additional improvements in photosynthetic WUE through osmoregulation and reduction of midday radiation interception respectively. Photosynthetic NUE was decreased by H₂O-stress in that control levels of total nitrogen, foliar chlorophyll, and RuBPCase activities were maintained even after mean diurnal levels of A had fallen to less than 50% of the control level. We conclude that field-grown sunflower manages a trade-off between photosynthetic WUE and NUE, increasing use efficiency of the scarce resource while decreasing use efficiency of the abundant resource.     

Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance. Received: 31 March 1996 / Revision received: 2 July 1997 / Accepted: 18 July 1997