Ivana Macháčková

Transgenic tobacco plants with T-DNA phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.

Free auxin level and inheritance of introduced markers in tobacco transformed by binary vector eased on A4 Ri plasmid

Tobacco cv. Mamont was transformed by leaf disc method with A.tumefaciens C58C1 (pRiA4b) (pGA472). Transformed leaf segments were cultivated on medium with kanamycin as a selection agent, but without phytohormones. Transformed tissues grew as roots which later regenerated plants. Kanamycin in the medium ensured that only doubly transformed tissues, which contain in their genome both pRi T-DNA as well as pGA472 chimeric kanamycin resistance gene were recovered. Regenerated plants showed clear-cut morphological deviations, but only a slight increase of the auxin content. Kanamycin resistance in the progeny segregated in the Mendelian ratio 3: 1 or 2: 1.

Characterization of phosphate absorption in maize root cortex segments

Uptake of phosphate ions by 1 mm segments of isolated maize root cortex layers was studied. Cortex segments (from roots of 8 days old maize plants) absorb phosphate ions from 1 mM KH2PO4 in 0.2 mM CaSCO4 at the average rate of 34.3 ±3.2 μg Pi g−1 (fr. m.) h−1,i.e. 0.35± 0.02 μmol Pi g−1 (fr. m.) h−1. Phosphate uptake considerably increases after a certain period of “augmentation”,i.e. washing in aerated 0.2 mM CaSO4. This increase is completely blocked by the presence of 10 μg ml−1 cycloheximide.The relation of uptake rate to phosphate concentration in the medium was shown to have 3 phases in the concentration range of 0.02 - 40 mM. Transition points were found between 0.8–1 mM and 10–20 mM. Following Km and Vmax values were found: Km[mM] : 0.37 - 3.82 - 27.67 Vmax[μg Pi g−1 (fr. m.) h−1] : 3.33 - 39.40 - 66.67We have found no sharp pH optimum for phosphate uptake. It proceeds at almost constant rate till pH 6.0 and then the uptake rate drops with increasing pH. At low phosphate concentrations (1 mM) the lowest uptake rate was found at 5 and 13 °C, while the uptake is higher at 5 °C than at 13 °C at phosphate concentrations higher than 1 mM. At these concentrations uptake rate at 35 °C is lower than at 25 °C.Phosphate uptake considerably decreased in anaerobic conditions. DNP and iodoacetate (0.1 mM) completely blocked phosphate uptake from 1 mM KH2PO4, while uptake from 5 and 10 mM KH2PO4 was left unaffected by these substances. The inhibitors of active - SH groups NEM and PCMB inhibited phosphate uptake: 10−3 M NEM by 81.6%, 104 M NEM by 42% and 10−4 M PCMB by 42%.

Comparison of the effect of some phenolic compounds on wheat coleoptile section growth with their effect on iaa-oxidase activity

P-coumaric acid (HCA), 2,4-dichlorophenol (DCP) and resorcionol acted as cofactors for IAA-oxidase isolated from young wheat plants. Ferulic acid (FA) and 3,4-dihydroxybenzoic acid (DHBA) induced a lag phase prior to IAA oxidation. HCA, FA (0.2-1 mg ml-1) and DCP (0.03-1 mg ml-1) strongly inhibited wheat coleoptile section growth. DHBA (0.01-1 mg ml-1) slightly stimulated it and resorcinol was without effect. HCA inhibited IAA-induced growth of coleoptile sections and FA stimulated it at low IAA levels and inhibited it at higher ones. DHBA, DCP and resorcinol did not affect IAA-induced growth of coleoptile sections.AbstractKyselina p-kumarová (HCA), 2,4-dichlorfenol (DCP) a resorcin působilyin vitro jako kofaktory IAA-oxidasy isolované z mladých rostlin pšenice. Kyseliny ferulová (FA) a 3,4-dihydroxybenzoová (DHBA) indukovaly před oxidací IAA lag fázi. HCA a FA (0,2-1 mg ml-1) a DCP (0,03-1 mg ml-1) silně inhibovaly růst segmentů pšeničných koleoptilí. DHBA (0,01-1 mg ml-1 jej mírně stimulovala a resoroin jej téměř neovlivnil. HCA brzdila IAA-indukovaný růst segmentů koleoptilí. FA jej při nízkýoh koncentracích IAA stimulovala, ale při vyšších koncentracích IAA brzdila. DHBA, DCP a resorcin neměly na IAA-indukovaný růst segmentů koleoptilí žádný vliv.

The effect of new male sterilants: Exo-3,4-methano-L-proline and cis-α-(carboxycyclopropyl)glycine on the IAA- and ACC-induced ethylene formation ia wheat coleoptile segments

The male sterilants exo-3,4-methano-L-proline (MP) and cis-α-(carboxycyclopropyl)glycine (CCPG) were not converted to ethylene, eitherin vitro or in wheat coleoptile segments. Neither of these substances was able to affect IAA- and ACC-induced ethylene biosynthesis in wheat coleoptile segments to such an extent that it could explain their effect ay male sterilants.

Higher flower bud formation in haploid tobacco is connected with higher peroxidase/IAA-oxidase activity, lower IAA content and ethylene production

Haploid tobacco plants (cv. Samsun) form inflorescences with a larger number of flowers than diploid plants. Leaves of haploid plants were shown to have lower free IAA level (by 40 %), higher peroxidase (by 160 %) and IAA-oxidase (by 70 %) activities and produce less ethylene (by 25 %) than leaves of corresponding diploid plants. The increase of peroxidase activity in haploids was due to the increase in the activity of the cathodic isozyme which is known to have high IAA-oxidase activity. It is proposed that higher peroxidase/IAA-oxidase activity in haploid plants may take part in IAA catabolism, at least duringin vitro culture of haploid explants. Lowered IAA level and ethylene production may then be directly correlated with a larger number of flower buds; as a higher IAA level is generally considered to act as a background inhibitor of flowering.

The effect of phenylacetic acid on ethylene formation in wheat seedlings

Phenylacetic acid (PAA) was found to induce ethylene formation in wheat coleoptile segments. In its most effective concentration (0.5 mM) PAA was by approximately 60 % less active than 0.1 mM indole-3-acetic acid (IAA). PAA-induced ethylene formation was stimulated with 0.1 mM L-methionine by 24 % and totally inhibited by 2.5 and 5 μ gml-1 aminoethoxyvinylglycin (AVG) and 10 μg ml-1 cycloheximide. Cyoloheximide in lower concentration (5 μg ml-1) and actinomycin D (10 μg ml-1) inhibited PAA-induced ethylene formation by 50 % and 40 %, respectively.After the simultaneous addition of PAA and IAA ethylene formation was by 35 % lower than in the presence of IAA itself. Further, the coleoptile segments preincubated in IAA and then incubated in PAA solution produced by 35 % less ethylene than those incubated in plain buffer after preincubation in IAA. Quite the opposite effect was found when the segments were preincubated in PAA and then transferred into IAA solution. This treatment resulted in 70 % stimulation of ethylene formation over segments preincubated in PAA and incubated in buffer.

The effect of indole-3-Acetic acid on ethylene formation in wheat seedlings

Isoperoxidase B 1 isolated from winter wheat (Triticum aestivum L., cv. Jubilar) seedlings was shown to catalyze ethylene formation from α-keto, γ-methylmercaptobutyric acid (KMBA). In the presence of Mn2+, indole-3-acetic acid (IAA), andp-coumaric acid, the kinetics by isoperoxidase B 1 catalyzed conversion of KMBA into ethylene and other products was similar to that of IAA oxidation. The reaction rate was therefore controlled by IAA through its electrondonating properties.Exogenous IAA induced ethylene formation in the segments of etiolated wheat coleoptiles. IAA-induced ethylene production was enhanced by L-methionine and mitomycin C. Aminoethoxy-analogue of rhizobitoxine, ferulic acid, sodium benzoate, cycloheximide and actinomyoin D exhibited significant inhibitory effects. These data indicate that the overall reaction mechanism in coleoptile segments involves RNA and protein synthesis.The site of IAA action is not specific; 2,4-dichlorophenoxyacetic, α-naphthylacetic and indole-3-butyric acids, respectively, possessed comparable inductive effect as IAA. Indole-3-propionic acid, indole, L-tryptophan and glucobrassicin had only low inductive efficiency, and moreover indole and L-tryptophan slowed down IAA-induced ethylene formation.

The effect of plant hormones on phosphate uptake and translocation in maize roots

In short-term (1 h) uptake experiments GA3(10-5M) stimulated Pi uptake into maize root cortex cells by 28.7 %, Ethrel (10-3M) inhibited it by 18.5 % and BA, IAA, and ABA were inactive. In long-term (5 h) experiments ABA remained inactive, GA3 lost its stimulatory effect, and BA (5. 10-6M), IAA (10-4 -10-5M), and Ethrel (10-3 -5. 10-4M) decreased Pi uptake. When the hormones were present only during 3 h preincubation (“augmentation”) period ABA was inactive, GA3 slightly raised and BA, IAA, and Ethrel slowed down subsequent Pi uptake.BA(10-7 –10-5M) decreased xylem sap volume flow and Pi translocation. ABA in all tested concentrations (10-8 –10-5M) reduced exudation rate and Pi translocation, its effect declining with time. IAA effect strongly depended on concentration used and on application time and varied from strong inhibition to moderate stimulation of both volume flow and Pi translocation. GA3 (10-7M) slightly stimulated xylem volume flow but inhibited phosphate translocation. Ethrel (10-4 and 10-5M) increased both parameters, but Pi transloeation much more than volume flow. IAA, BA, and ABA influenced volume flow and P transloeation to the same extent leaving Pi concentration in the xylem sap unchanged. GA3 and Ethrel influence Pi concentration in the xylem sap and it is thus probable that these hormones regulate release of phosphate ions into the xylem sap.

The effect of some micronutrients and heavy metals on phosphate absorption by maize root cortex segments

The effect of salts (nitrates, chlorides, and sulfates) of microelements, Cd2+, Ni2+, and Co2+ and the effect of boric acid and ammonium molybdate on phosphate uptake by maize root cortex segments were tested.Higher concentration (0.1 mM) of Cu2+ salts caused enhancement of phosphate efflux to the extent that efflux was higher than influx.Inhibitory action on phosphate uptake by maize root cortex segments was exerted by following salts: 0.01 mM Cu2+ salts (20–30% inhibition), 0.5 mM ZnSO4 (9.7%), 0.5 and 0.05 mM ZnCl2 (34.3% and 20.8%), 0.1 mM salts of Cd2+, Ni2+, Co2+ (35–78%).1 mM FeSO4 had significant stimulatory effect (92%) on phosphate uptake. Much weaker stimulatory effect was exerted by 1 mM FeCl3 (14%), 0.05 mM ZnSO4 (9.6%), 0.005 mM ZnCla and ZnSO4 (8.4 and 18.5%) and 0.001 mM CdCl2 and CdSO4 (20.8 and 12.4%).All other tested salts-salts of Mn2+ (0.1 and 0.01 mM), 0.01 and 0.001 mM salts of Co2+ and Ni2+, 0.001 mM salts of Cu2+, 0.001–10 mM boric acid, and 0.001–0.1 mM ammonium molybdate left phosphate uptake unaffected.