A simple procedure for the detection of extracellular plant proteolytic enzymes using insoluble dye stained gelatin substrates incorporated into an appropriate culture medium is described. Extracellular proteinases produced by the tested plant cells (callus culture and cell suspension) hydrolyzed the substrates and dyed peptide fragments were released. Dyed zones around and under the proteinase-producing callus cultures were formed on the agar medium. Similarly, coloration of the culture media using proteinase-producing cell suspensions was observed.

Genetic tumour tissues of Nicotiana glauca (Grah.) × N. langsdorffii (Weinm.), which grow on auxin and cytokinin-free medium, were incubated with [14C]-/[3H]-adenine or [3H]-hypoxanthine to investigate cytokinin biosynthesis. Adenine was supplied to tissues of two different ages (2- and 3.5-week-old) for 8, 24 or 30 h. The uptake was over 91.0 % (of "supplied radioactivity") by 2-week-old tissues as compared to around 50.0 % uptake by 3.5-week-old tissues. Incorporation into cytokinins could not be detected. While unmetabolized adenine accounted for only about 24.0 and 13.4 % of "extracted radioactivity" (following 8 and 30 h incubation, respectively) in 2-week-old tissues, relatively higher levels, i.e. 36.0 and 34.5 % (following 8 and 24 h incubation, respectively) were present in 3.5-week-old tissues. The metabolites formed were adenosine and its nucleotides (4.5 - 16.5 % and 37.4 - 60.2 % of the extracted radioactivity, respectively). Hypoxanthine was supplied to 3.5-week-old tissues for 8 and 24 h. While the uptake was low (<28.0 % of supplied radioactivity), the major proportion of extracted radioactivity was due to unmetabolized hypoxanthine (79.8 % and 85.9 % after 8 and 24 h incubation periods, respectively); the minor metabolites were inosine and adenosine (both <0.5 %) and their nucleotides (< 3.5 %). Radioactivity incorporation into cytokinins from hypoxanthine was not detected. Thus in the present investigations precursor incorporation from either adenine or hypoxanthine into cytokinins could not be demonstrated. It is possible that this may be due to slow rate of cytokinin turnover in these tissues.

Several pieces of research suggest that leaf liquid-path conductance (K1) may be smaller in evergreen species (EG) than in deciduous species (DC), coexisting in tropical dry forests. This was investigated in four DC and two EG in a tropical dry forest in Venezuela. The K1 (kg m-2 MPa-1 s-1) were consistently lower in EG (8.4 × 10-5) than in DC (12 to 20 × 10-5). Differences in water potential (ΔΨW) inside the leaves were calculated using K1 values in connection with maximum transpiration rates measured in the field. It was found that ΔΨW was about 1.9 MPa in EG, intermediate for one DC (Pithecellobium dulce, 1.7 MPa) and minimum for other DC (1.0 to 1.3 MPa).

The effect of cytokinins (CKs) and K+ on stomatal behaviour in darkness were studied in the root hemiparasite Melampyrum arvense before (the preparasitic stage) and after attachment to the host (Capsella bursa pastoris L. Med.). The solutes were applied with xylem stream. The stomatal apparatus of the attached hemiparasite was insensitive to externally supplied CKs and K+. Contrary to this finding, the stomatal aperture of hemiparasite in the preparasitic stage increased to about 25, 40 and 69% of the value obtained in light, respectively, after treatment with 200 mM KCl and 10-5 M zeatin riboside ([9R]Z), applied separately or together. CKs influenced K+ transport. The treatment with KCl and [9R]Z,separately or together, increased the content of K+ in guard cell pairs to about 32, 46 and 79 % of the value obtained in light, respectively. Other CKs had a smaller effect (45 - 16 %) in comparison with that of [9R]Z; isopentyladenine was nearly inactive.

We have examined the toxicity of ethanol in tissue culture of the apple rootstock 'Jork 9'. During proliferation through axillary branching, 0.2% (v/v) ethanol slightly stimulated proliferation whereas significant inhibition occurred at concentrations of 0.4 % or higher. In adventitious root formation, significant inhibition occurred at concentrations of 0.1 % or higher. The effect of ethanol was stage-dependent: during the induction period (i.e. from 24 to 72 h after the start of the rooting treatment), there was little or no inhibition. During autoclaving, ethanol evaporated to ca. 50 %.

Standard methods of isolation of chromatin histones from Arabidopsis thaliana yield strongly degraded proteins when applied to plants grown from seeds in axenic liquid media. For isolation of undegraded histones from Arabidopsis grown in liquid media we used extraction with guanidine hydrochloride followed by selective binding of histones on BioRex 70 resin in the batch system. The quality of obtained proteins is confirmed by SDS polyacrylamide gel electrophoresis.

The mechanisms of saccharose and sorbitol transport in Prunus persica leaves were investigated in plasma membrane vesicles purified by aqueous 2-phase partitioning and equilibrated in pH 7.5 buffer containing K+. The imposition of an artificial proton motive force energized an active uptake of both saccharose and sorbitol. The maximum uptake rate of saccharose was 2.5 times higher than that of sorbitol. Saccharose and sorbitol uptake exhibited saturation kinetics suggesting they were carrier-mediated. Apparent Km for the saccharose and the sorbitol uptake were 0.36 and 0.67 mM, respectively. Active absorption of saccharose was completely inhibited by a non-permeant thiol reagent, PCMBS, contrary to sorbitol absorption. These results suggested that saccharose and sorbitol were transported at least by two different carriers.

Suspension-cultured rice cells growth was markedly inhibited and ammonium content increased when rice cells were deprived of phosphate. When rice cells were cultured at increasing concentrations of ammonium chloride, ammonium content increased, however, no significant inhibition of cell growth was observed. Addition of D-arginine, an inhibitor of putrescine biosynthesis, resulted in a complete recovery of growth in rice cells under phosphate deprivation, but did not decrease the content of ammonium. Our results indicate that the growth inhibition induced by phosphate deprivation is not associated with ammonium accumulation.

Triazine (0.6 mM) quenched the chlorophyll fluorescence emission at 686nm, selectively reduced the biosynthesis of linolenic acid (C18:3), lowered the ratio of unsaturated/saturated fatty acids in thylakoid membranes and percentage of total glycerides in the extracted oils, and extremely enhanced ethylene production by seedlings. These effects were partly reversed by the addition of 100 g m-3 of vitamins (thiamine > nicotinic acid >pyridoxine).

In order to clarify the role of endogenous growth inhibitors A-2α and A-2β in a dwarf pea plant, red light (emission peak 657 nm) treated, 9-d-old seedlings of dwarf pea (Pisum sativum L. cv. Progress No. 9) were transferred to darkness, and the resulting changes in growth rate and concentrations of A-2α and A-2β were monitored. The growth rate of the epicotyls increased, and the concentration of the inhibitors in the epicotyls decreased, according to sigmoidal time courses. The relationship between the logarithms of the concentration of the inhibitors and the corresponding growth rate was linear. These results suggest that A-2α and A-2β, may play an important role in the growth recovery process of the dwarf pea cultivar after termination of red light irradiation.

The response of Brassica carinata hybrids and their parents to moisture stress at different growth stages was studied. B. carinata 226 was found to be susceptible to stress at pre-flowering and post-flowering stages while B. carinata 241 at flowering stage. Neither the changes in stomatal conductance nor in chlorophyll content could fully explain the reduction in net photosynthetic rate (P_N) induced by stress. B. carinata 241 had higher leaf water potential (ψ_w) although, it had lower P_N compared to B. carinata 226. Both the parents had lower P_N as well as leaf ψ_w. The stress response of PN in hybrids followed that in their respective female parents.

Seedlings of the salt sensitive wheat cultivar C-306 evolved more ethylene than the salt tolerant cultivar Kharchia-65 under different levels of both chloride- and sulphate-dominated types of salinity. Pre-sowing seed soaking treatments with kinetin, gibberellic acid and to a lesser extent indole-3-acetic acid alleviated salt stress effects as apparent from seedling dry mass. Treated seedlings also evolved more ethylene both under saline and non-saline conditions. Ethrel did not affect seedling growth as well as ethylene production. Abscisic acid inhibited seedling growth and ethylene production under both types of salinity.

The role of plant antioxidant system in water stress tolerance was studied in three contrasting wheat genotypes. Water stress imposed at different stages after anthesis resulted in a general increase in lipid peroxidation (LPO) and decrease in membrane stability index (MSI), and contents of chlorophylls (Chl) and carotenoids (Car). Antioxidant enzymes like glutathione reductase and ascorbate peroxidase significantly increased under water stress. Genotype C 306, which had highest glutathione reductase and ascorbate peroxidase activity, also showed lowest LPO and highest MSI, and Chl and Car contents under water stress in comparison to susceptible genotype HD 2329, which showed lowest antioxidant enzyme activity as well as MSI, Chl and Car contents and highest LPO. HD 2285 which is tolerant to high temperature during grain filling period showed intermediate behaviour. Thus, the relative tolerance of a genotype to water stress as reflected by its comparatively lower LPO and higher MSI, Chl and Car contents is closely associated with its antioxidant enzyme system.

Activity of Cu2+-diamine oxidase (DAO; E.C.1.4.3.6.) was measured in Vigna radiata (L.) Wilczek cultivars K851, MH8320 and Pusa Baisakhi in light and in dark during ontogeny of seedlings. DAO activity was always the highest in cv. K851. In both light and dark grown seedlings maximum DAO activity was detected on day 2 after germination. Thereafter, in light grown seedlings it declined consistently upto non-detectable levels. In dark, DAO activity was higher than in light and it had the second maximum on day 7 following a similar declining pattern as observed for the light grown seedlings. The DAO activity was higher in a shoot apex alongwith leaves than in roots and shoot axis.

Maturation and germination of somatic embryos of hybrids A. alba × A. alba and A. alba × A. nordmanniana were followed by protein analysis of single embryogenic -suspensor masses (ESM) and analysis of storage protein accumulation during somatic embryo development. Very important step was one week pre-cultivation of ESM on medium with polyethylene glycol (PEG) and abscisic acid (ABA). Low osmotic potential of maturation medium and addition of ABA supported development of somatic embryo. Also partial drying of somatic embryo during following three weeks was needed for its normal development. In spite of morphologically fully developed, the somatic embryos were not physiologically ready for germination at least in terms of storage protein accumulation.

Uptake mechanisms for neutral amino acids were investigated by expression of mRNA isolated from seedlings of Ricinus communis L. in Xenopus laevis oocytes. After injection of mRNA from root, hypocotyl and cotyledon currents elicited by saccharose and neutral amino acids ranged from 0.3 nA up to 2 nA depending on the respective substrate and the source of mRNA. These currents were due to expression of low affinity uptake mechanisms and the KM values found for amino acid induced charge flow range from 1 to 2 mM. The abundance and/or the specificity of the expressed mechanisms differ in the various tissues. Currents of similar magnitude were recorded for alanine and glutamine with mRNA isolated from root, hypocotyl and cotyledons. Serine and proline induced currents after injection of mRNA from hypocotyl and roots, in case of α-aminoisobutyric acid (AIB) induced currents were generally small with mRNA from all tissues tested. In addition, differential sensitivity of glutamine and AIB uptake in the high affinity range was evident towards the amino acid analogue 2-chloro-aminophenoxybutyric acid which indicated an additional set of carriers operating in the micromolar concentration range. The results suggest that multiple transporters for neutral amino acids exist in various tissues of the plant differing in specificity of charge flow and in sensitivity towards the inhibitor 2-chloro-aminophenoxybutyric acid.

Plants of Calamagrostis villosa were cultivated in nutrient solution alone or in association with a vesicular-arbuscular mycorrhizal (VAM) fungus Glomus etunicatum. They were supplied with two levels of inorganic phosphate (Pi; 0.1 or 1 mM) and with or without organic phosphate (dinatriumphenylphosphate, Po; 1 mM). Depression of growth and enhancement of root respiration of mycorrhizal plants in comparison with non-mycorrhizal plants were observed after 12 weeks of cultivation in a growth cabinet. Root colonization was not influenced by the higher phosphorus availability in contrast to the extraradical mycelium (ERM). The lengths of ERM hyphae both attached to the root surface and in the substrate were decreased substantially by higher phosphorus supply, irrespectively of its form.

Alfalfa (Medicago sativa L.) cell suspension cultures of Verticillium albo-atrum resistant and susceptible genotypes were established from leaf callus tissues. Treatment of cultures with conidia and heat-released elicitors of V. albo-atrum induced a large increase in the activity of phenylalanine ammonia-lyase, only in the cells of the resistant genotypes with a maximum after 12 h. In co-cultivation with the fungal conidia and resistant cell lines, the production of spores were inhibited.

The immature zygotic embryos of reciprocal maize hybrids (CHI-31 x GF1 and CHI-31 × GE2) were used as the initial material for induction of somatic embryogenesis in vitro. Histological analysis of somatic embryogenesis revealed high developmental variability. The arising formations were classified into 5 groups: A) somatic embryos phenotypically similar to zygotic embryos, B) polyembryos, C) formations with radicle but without meristematic plumule, D) formations with radicle without differentiated plumule, and E) formations with plumule without radicle. The formatioms A and B regenerated directly into plants. Plant regeneration from formations E required preculture on the rooting medium. Formations C and D failed to develope into plants possibly because of early loss of meristematic cell character during the embryo axis differentiation. The reverse sequence of radicle and plumule differentiation in somatic embryos in comparison with zygotic ones was noted. The epigenetic character of the scutellum, coleoptile, coleorhiza and leaves primordia development was discussed.

Sugar beet was grown in pots and the transport of 6-deoxy-d-glucose (6-dg) and of 2-aminoisobutyric acid (AIB) into leaf and root tissue segments was examined. The uptake of 6-dg into both tissues displayed two kinetic components, one apparently of diffusional nature, the other saturable (half-saturation constant 0.22 mM in leaves, 0.10 mM in roots; limiting rate 0.37 and 0.10 nmol mg-1 (fresh mass) h-1 for leaves and roots, respectively). It was suppressed by d-glucose and inhibited by diethyl-stilbestrol (DES), suloctidil (SUL) and 2,4-dinitrophenol (DNP), but little by D2O and not at all by sodium vanadate. It showed a temperature optimum at 30 °C and a pH optimum at 6.0 - 6.5 in both tissues. The uptake of AIB was also two-componental but even the specific component had an immeasurably high half-saturation constant although it was inhibited by high concentrations of AIB as well as by DES, SUL, DNP and D2O. However, vanadate stimulated the uptake by as much as 40% in both leaves and roots. The temperature optimum in leaves lay at 30 °C while in the roots no maximum was present; its pH optimum was at 5.5 in leaves and again no maximum was observed in roots. Both transport systems were unaffected by 0.1M NaCl or 0.1 M KCl and by the presence of cytokinin in the incubation vessel. However, spraying with the cytokinin N6-(m-hydroxybenzyl) adenosine decreased both the half-saturation constant and the limiting velocity of 6-dg uptake slightly in the leaves and substantially in the roots. Spraying had an insignificant effect on the uptake of AIB.

Age-induced changes in 1) nocturnal and diurnal acidity fluctuations that coincide with the ongoing environmental conditions, 2) the build up of abscisic acid (ABA) in plant roots and leaves during sunrise, midday, and sunset in all growing stages, 3) the changes in phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) activities as key enzymes of the photosynthetic pathways of C3 and CAM, 4) leaf water potential (ψ1), and 5) Km and Vmax for PEPC to express its activity and affinity, were studied in Mesembryanthemum nodiflorum during transition from C3 to CAM mode of CO2 fixation. The acidity during sunset in mature stage was higher than in earlier stages and reflected the impact of environmental conditions on physiological and metabolic changes. Moreover, the higher acidity during sunrise and sunset was observed during the senescence than the mature stage; this might be due to CO2 release and oxygen intake during senescence induced ethylene formation that lead to increased malic acid formation. The ABA concentration was high in M. nodiflorum leaves, but stomatal closure was insensitive to elevated ABA concentrations recorded. Vmax of PEPC, Km, and the affinity of PEPC during later stages indicated the ability of PEPC to fix CO2 taking up at night in CAM cycle of M. nodiflorum. Less affinity during sunrise indicated inhibitory effect of malate on PEPC during the release of CO2. The second peak of PEPC activity before sunset caused CO2 fixation. The RuBPCO was inactive at night. Slight increase in ABA during sunset, and night drop in air temperature and increase in relative humidity reduced markedly transpiration rate without decreasing ψ1.

Indoor experiments demonstrated that allelopathic potential of rosette and flowering plants of qort is an important factor explaining the growth reduction of its associated species. Aqueous tissue extracts of flowering plants exhibited strong inhibitory effects on the germination percentage and radicle growth rate of the tested species as compared with those of vegetative plants. Under laboratory conditions, this inhibition was in agreement with toxicity assessments of soil samples collected from the rhizosphere of T. resupinatum L., where shoot and root dry mass of the tested species were significantly reduced. Detoxification of allelochemicals by presence of activated carbon can eliminate the inhibitory effects of the different extracts. This technique clarifies the occurrence of allelopathic interference by qort on seed germination and seedling growth, and hence suspects the allelopathic potential of qort in the growth reduction of associate species under field conditions along with competition.