Stanislav Procházka

Activation of caspase-like proteases and induction of apoptosis by isopentenyladenosine in tobacco BY-2 cells

Abstract. The effects of isopentenyladenosine (iPA) on tobacco (Nicotiana tabacum L.) BY-2 cells were examined. The number of BY-2 cells decreased in a time- and concentration-dependent manner after being exposed to micromolar concentrations of iPA. This decrease was mainly due to a loss of cell viability, since no substantial changes in cell cycle progression were revealed by flow-cytometric analysis. Dying cells exhibited the typical morphological and biochemical hallmarks of apoptosis, including cell shrinkage, chromatin condensation, and degradation of nuclear DNA to nucleosomal size fragments. Caspase-1-like and caspase-3-like proteases also became activated, the former being dominant. Inhibitor-sensitivity studies revealed that although synthetic caspase inhibitors failed to prevent cell death they markedly reduced cell death in tobacco BY-2 cells, Ν-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, a specific inhibitor for caspase-1, being the most effective. Our results indicate that caspase-like proteases, and particularly caspase-1-like protease, might be critically implicated in iPA-induced apoptosis of BY-2 cells. Finally, the outcome of inhibiting adenosine kinase by 4-amino-3-iodo-1(β-D-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine revealed that intracellular phosphorylation of iPA is required for its cytotoxicity to develop.

Auxin and cytokinins in the control of apical dominance in pea: A differential response due to bud position

Summary Twenty one-day-old pea plants were used as a convenient model to investigate the release of axillary buds from apical dominance with respect to their serial position and the timing of events employed as indicators of growth changes. These were recorded in the second and fifth buds from the base, possessing the highest growth potential. The decapitation and consequent loss of apical dominance led to an increase in fresh weight of the fifth bud already after 24 h, with the similar but delayed response of the second bud. The rate of 32 P incorporation into buds had a similar pattern but appeared much earlier, as soon as 6 h after application. Decapitation significantly decreased the indole-3-acetic acid level in both buds already after 6 h. A rise in the cytokinin content was observed already after 6 h following decapitation, with the highest increment in isopentenyladenosine and zeatin riboside. IAA treatment of the decapitated plants simulated to various degrees an intact apex and eliminated the effects of decapitation. These results are in favour of both auxin and cytokinin involvement in the control of apical dominance.

Involvement of auxin and cytokinins in initiation of growth of isolated pea buds

Axillary buds from the second primary scale excised from 21-day-old pea(Pisum sativum L. cv. Vladan) plants were used as a modelsystem for studying the release of buds from apical dominance. The isolatedbudswere transferred onto basal medium with or without a supplement of growthregulators and cultivated up to 24 h in short-term and up to 4weeks in long-term experiments. In both sets of experiments endogenous IAA,cytokinins and the uptake of labelled zeatin were analysed. The development ofbuds was monitored by image analysis, estimation of their weight, and byanatomical studies. Generative meristems were found in isolated axillary budsalready in 21-day-old plants at the beginning of the experimental period. Theonset of bud growth was recorded as soon as 2 h after the budexcision by image analysis. The bud growth was accompanied by a rapid transientincrease of the endogenous IAA level within the first 2 h,followedby an increase of iPA within 24 h. The uptake of the exogenouscytokinin ([3H]Z) reached its peak between the 6 and 8hafter the release from apical dominance. The cytokinin analyses of bothshort-term and long-term bud cultures revealed the increase of free cytokininsand their glucosides, indicating de novo synthesis ofcytokinins in the buds themselves.

The transformation of pea (Pisum sativum L.): applicable methods of Agrobacterium tumefaciens-mediated gene transfer

Three methods of transformation of pea (Pisum sativum ssp. sativum L. var. medullare) were tested. The most efficient Agrobacterium tumefaciens-mediated T-DNA transfer was obtained using embryonic segments from mature pea seeds as initial explants. The transformation procedure was based on the transfer of the T-DNA region with the reporter gene uidA and selection gene bar. The expression of β-glucuronidase (GUS) in the regenerated shoots was tested using the histochemical method and the shoots were selected on a medium containing phosphinothricin (PPT). The shoots of putative transformants were rooted and transferred to non-sterile conditions. Transient expression of the uidA gene in the tissues after co-cultivation and in the course of short-term shoot cultivation (confirmed by histochemical analysis of GUS and by RT-PCR of mRNA) was achieved; however, we have not yet succeeded in proving stable incorporation of the transgene in the analysed plants.

Translocation of growth regulators from roots in relation to the stem apical dominance in pea (Pisum sativum L.) seedlings

Since it has been found that roots are a source of cytokinins [2], [8] and gibberellins [1,7] and that exogenous applications of cytokinins and gibberellins can promote bud growth [5], [9] several authors have studied the relationships between the transport of growth regulators from roots and the development of lateral buds [3]. The role of growth regulators synthetized in roots and translocated into the above-ground part of plants in relation to the apical dominance of the stem has not yet been completely explained. The condition of a bud releasing itself from a correlative inhibition is changing very quickly and involves two stages, viz. (a) the release from the correlative inhibition and (b) the subsequent growth of the lateral bud [6].

Stimulation of 14C-Abscisic Acid Transport by IAA and Sucrose in Pea Epicotyls

Summary The effects of IAA and sucrose upon the transport of 14C-ABA was studied in epicotyl segments of etiolated pea seedlings.It was found that 14C-ABA moved more intensively in the basipetal direction.Both IAA and sucrose promoted the basipetal transport of 14C-ABA.This phenomenon is discussed with regard to the possible nature of ABA transport.

The effect of cycloheximide on IAA-stimulated transport of 14C-ABA and 14C-sucrose in long pea epicotyl segments

The effect of cycloheximide (CH) on the indol-3yl-acetic acid (IAA)-stimulated transport of 14C-labelled abscisic acid (ABA) and 14C-labelled sucrose was studied in 110 mm long pea epicotyl segments. IAA application resulted in elongation growth of the segments. This effect was decreased by CH treatment which also reduced [14C] ABA and [14C] sucrose accumulation in the growing apical part of the segments. A reduction in [14C] IAA uptake and in protein synthesis in this part of the segments was also observed. The simultaneous inhibition of protein synthesis and reduction of [14C] ABA and [14C] sucrose transport suggests that IAA can stimulate the transport of ABA and sucrose through a protein synthesis-based elongation growth.

Morphoregulative Effects of Phenylacetic Acid in Pea Seedlings (Pisum sativum L.)

Summary The effect of indole-3-acetic acid and phenylacetic acid upon the thickening growth of epicotyl, growth of lateral buds, and attraction of benzyl-[8-14C]-adenine from roots was studied in 5 d-old etiolated pea seedlings. The morphoregulative ability of phenylacetic acid in apical dominance was similar to that of indole-3-acetic acid, however, it is necessary to use much higher concentrations of phenylacetic acid than of indole-3-acetic acid. Phenylacetic acid applied exogenously to stumps of decapitated plants was able to attract benzyl-[8-14C]-adenin from roots similarly as indole-3-acetic acid. However, in contrast to indole-3-acetic acid it does not prevent the flow of benzyl-[8-14C]-adenine into lateral buds and even promotes it.

The uptake and metabolism of 3H-benzylaminopurine in tobacco (Nicotiana tabacum L.) and cucumber (Cucumis sativus L.) explants.

The uptake and metabolism of 3H-benzylaminopurine(3H-BAP) were studied in explanted stem pith andleaves of tobacco and in the hypocotyls and cotyledonsof cucumber. The explants were kept for 2, 5, 8 and 20h on MS medium with 0.8 mg.l−1 2,4-D,0.5 mg.l−1 BAP and 13.2 mg.l−1 aspartic acid(induction medium) with or without 3H-BAP and14C-sucrose. The highest uptake of 3H-BAPwas observed in tobacco leaves and cucumbercotyledons. The major metabolite in both species was3H-benzylaminopurine riboside (3H-BAPR). Thehighest level was found in explanted cucumbercotyledons after 20 h in culture, the lowest inexplanted tobacco stem pith. Intensive 7-glucosylationof 3H-BAP was observed in explanted tobaccoleaves after 20 h in culture, where the levels of7-glucoside of 3H-BAP and of free 3H-BAPwere equal. To study the morphogenic effect of growth regulators(BAP and 2,4-D), the explants were subcultured aftershort-term induction (20 h) to MS medium without anygrowth regulators. In most cases, incubation of 20 hon induction medium was sufficient to induce therespective morphogenesis. Cucumber hypocotyl andtobacco stem pith explants formed a callus on theirbasal end. Root formation was observed on explantedcucumber cotyledons and shoot formation on tobaccoleaves. Long-term culture (3 weeks) of tobacco leaveson induction medium led to the formation of callus andglobules. The microscopic analysis of globulesindicated the presence of meristematic and tracheidalcells.

Release of cotyledonary shoots of pea (Pisum sativum) seedlings from inhibition as related to endogenous zeatin and ethylene and exogenous IAA and benzyladenine

This paper deals with apical dominance using a dicotylar model obtained after decapitation of pea seedlings with two shoots — one dominant and the other inhibited. When the dominant shoot was decapitated the inhibited one is released from inhibition and after 24 to 72 h begins to grow. However, the levels of trans-zeatin and production of ethylene increase within 4 and 6 hours respectively after release from inhibition, and within an interval of 72 h the levels of both phytohormones begin gradually to decrease. This indicates that also in this model, the release from apical dominance is associated with an increase in the level of cytokinin zeatin and, thereafter, also with an increased production of ethylene. If indolyl-3-acetic acid (IAA) is applied on the decapitated main stem after decapitation of the dominant shoot, the growth of the initially inhibited one is very strongly retarded; if, however, IAA is applied on the decapitated dominant shoot, this inhibition is significantly weaker. This means that the inhibiting effect of IAA on the inhibited shoot originates to a greater degree from the main stem rather than from the dominant shoot. The effect of benzyladenine (BA) is transferred equally from the decapitated main stem and from the decapitated dominant shoot because the initially inhibited shoot begins to grow as well as also other shoots from serial cotyledonary buds.