Marek Klemš

Evaluation of biological activity of new synthetic brassinolide analogs

The responses of plants to exogenous treatment with new synthetic brassinosteroids (BRs) were assessed and compared with the activity of natural 24-epibrassinolide (24-EPI). Morphological experiments on plants of pea and flax showed that the boundary between stimulatory and inhibitory concentrations of individual BRs and 24-EPI used is very narrow and differs also with the plant species. Moreover brassinosteroids can exhibit effects similar to various other plant hormones. This was proven also in our experiments, where auxin, anti-auxin and cytokinin like effects were achieved by BRs application. One of the explanations of the different morphological effects could be the influence of brassinosteroid application on the level of endogenous hormones. There are changes in the levels of indole-3-acetic acid, 6-benzylaminopurine, trans-zeatin and dihydrozeatin in rape and wheat plants caused by BR 4 and 24-EPI application, but there is no general trend explaining unequivocally their influence. The fact that all tested BRs significantly increased the dry weight accumulation in comparison with non-treated reference rape plants can be accounted for the known BRs characteristics to avoid biotic stresses.

Root and foliar uptake, translocation, and distribution of [14C] fluoranthene in pea plants (Pisum sativum)

Uptake of (14)C-labeled fluoranthene ([(14)C]FLT) via both roots and leaves of Pisum sativum seedlings and distribution of [(14) C] in plants by both acropetal and basipetal transport was evaluated. The highest [(14)C] level was found in the root base (≈270 × 10(4) dpm/g dry wt) and the lowest level in the stem apex (<2 × 10(4) dpm/g dry wt) after just 2 h of root exposure. For foliar uptake, the highest level of [(14)C] was found in the stem and root apex (both ≈2 × 10(4) dpm/g dry wt) (except for treated leaves), while the lowest level was found in the root base (<0.6 × 10(4) dpm/g dry wt).

Changes in cytokinin levels and metabolism in tobacco (Nicotiana tabacum L.) explants during in vitro shoot organogenesis induced by trans-zeatin and dihydrozeatin

The uptake and metabolism of trans-zeatin and/or dihydrozeatin, in correlation with cytokinin oxidase/dehydrogenase (CKX) and β-glucosidase activity, were studied in leaf segments derived from wild-type (WT) and transgenic (T) tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) during in vitro induction of shoot organogenesis. T explants harbored the maize gene Zm-p60.1β-glucosidase. Higher levels of shoot regeneration were observed on T explants in the early stages of cultivation. In WT explants, the content of cytokinin (CK)-O- and N-glucosides increased. In T explants, a higher content of Z-9-riboside and Z-9-riboside-5′-monophosphate and higher CKX activity during the early stage of cultures were found. A positive correlation was obtained for bioactive CK content and the organogenic response in T explants. Our results indicate a connection between the organogenic capacity of tobacco explants, metabolism of endogenous CKs and uptake of exogenous CKs from the cultivation medium.

Uptake, transport and metabolism of 14C-2,4-dichlorophenoxyacetic acid (14C-2,4-D) in cucumber (Cucumis sativus L.) explants

The uptake, distribution and metabolism of 2,4-D using 14C-labelled 2,4-dichlorophenoxyacetic acid (14C-2,4-D) was studied in isolated hypocotyl and cotyledon explants of cucumber (Cucumis sativus L.) in vitro. Cotyledons had a higher uptake capacity than hypocotyls; the uptake in cotyledons increased linearly up to 20 h, while in hypocotyls only up to 8 h. The distribution of 14C-activity in both organs was basipetal, but more pronounced in cotyledons. The 2,4-D taken up by cotyledons was metabolized very rapidly: only 7% of 14C-activity was associated with free 2,4-D after 20 h exposure. Hypocotyls metabolized 2,4-D more slowly: after 20 h 50% of the 14C-activity was still associated with free 2,4-D. After short incubation periods (2–5 h) 2,4-D-aspartate was a predominant metabolite, after longer incubation (8–20 h) 2,4-D-glucosyl ester prevailed. Roots or callus were formed on the base of cotyledons depending on the length of exposure to 2,4-D.

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.

The uptake and distribution of 14C-NAA in cotyledons of cucumber (Cucumis sativus L.) in vitro

1-naphtalene acetic acid (NAA) belongs to the group of auxin growth regulators and is widely used in in vitro cultures for the induction of morphogenesis or dedifferentiation. The morphogenic effects of NAA are closely related to its transport and accumulation as well as the interactions with other growth regulators (Davies et al., 1995; Coenen and Lomax, 1997). On the medium with NAA Van den Ende et al. (1984) induced the formation of flower buds on the tobacco thin tissue layers. Smulders et al. (1986) observed that the formation of flower buds depends on concentration of NAA in medium, its uptake and transport within the explant. The uptake of NAA into plant cell occurs mainly via free diffusion, whereas its export takes place mainly through membrane carriers (Delbarre et al., 1996). In the tobacco stem pith 14C-NAA is taken up intensively already within the first hours of exposure (Abbas et al., 1995). The taken up NAA is rapidly metabolized. Smulders et al. (1990) reported that as much as 80% of total 14C-NAA taken up was metabolized already after 6 hours. Aranda et al. (1984) have identified the forms of NAA conjugated with glucose or aminoacids. The selective uptake, transport and metabolism of growth regulating substances depend strongly on the physiological stage and the degree of differentiation of the explant (Abbas et al., 1995).