Jolanta Legocka

Role of spermidine in the stabilization of the apoprotein of the light-harvesting chlorophyll a-b-protein complex of photosystem II during leaf senescence process

Barley leaf discs maintained in dark accumulated a massive amount of putrescine (Put), lost chlorophyll and senescenced rapidly. At the same time RNase activity increased significantly. Exogenous spermidine (Spd) inhibited RNase activity, the loss of chlorophyll and degradation of the proteins from thylakoid membranes. Using SDS-PAGE and immunoblot analysis it was shown that spermidine was effective in the retardation of the loss of LHCPII observed in water-treated detached leaves. Analysis of PSII particles isolated from leaf fragments floated in water in the dark revealed the presence of Put, Spd and Spm. In spermidine treated leaves the level of this polyamine in photosystem II was above 5-fold higher than in control. The experimental findings obtained in this study provide evidence that applied spermidine interacts directly with thylakoid membranes so that they become more stable to degradation during senescence.

Role of polyamines in the cytokinin-dependent physiological processes II. Modulation of polyamine levels during cytokinin-stimulated expansion of cucumber cotyledons

The cucumber cotyledon expansion test was used as a model system to study a possible relationship between cytokinin and polyamines. When kinetin was applied to excised cotyledons incubated in the dark it caused a marked increase in the activity of arginine decarboxylase. As a result of ADC action, putrescine content also rose markedly, whereas the level of spermidine and spermine decreased. However, inhibition of putrescine biosynthesis with D-arginine did not affect cytokinin promotion growth. Applied alone, putrescine had no significant effect on growth. These results indicate that the large increase in putrescine content that derives from cytokinin treatment cotyledons is not essential for cytokinin-induced expansion of cotyledons. Addition of K+ and Ca2+ ions to the cotyledons incubated with cytokinin caused a marked reduction in the putrescine level and ADC activity.The higher level of putrescine (35 %) and spermine (62 %) bound to chromatin and the large increase (174 %) in spermidine content bound to ribosomes which derive from cytokinintreated cotyledons in relation to literature data can indicate that these polyamines may play an important role in gene expression during cytokinin-stimulated expansion of cucumber cotyledons.The inhibition of cytokinin effect, viz. enlargement of the cotyledons by inhibitors of spermidine biosynthesis, additionally suggessted a possible involvement of polyamines in cytokinin action.

The Role of Cytokinins in the Development and Metabolism of Barley Leaves III. The Effect on the RNA Metabolism in Various Cell Compartments During Senescence

Summary The degradation of RNA in excised barley leaves in darkness mainly concerned the 25 S and 18 S rRNAs, whereas the 23 S and 16 S rRNAs were. rehtively stable. The cytokinin prevented an increase in the RNase activity and caused an increase in the RNA level and in the incorporation of 14 C-uracil into the cytoplasmic rRNAs, their precursors and the low molecular RNAs. There was no incorporation of the precursor into the 23 S and 16 S RNAs. Changes in RNA resulting from the senescence and from the cytokinin treatment occurred mainly in the cytoplasmic and nuclear cell fractions; they were relatively small or non-existent in the chloroplast fraction. It is concluded that changes in chloroplast structure during senescence of barley leaves are rather secondary results of processes occuring in other cell compartments. Also the retarding effect of cytokinin on the chloroplast senescence seems to be rather indirect and resulting inter alia from a protection of the RNA and a stimulation of its synthesis in the cytoplasm and nucleus.

Dark-induced senescence of barley leaves involves activation of plastid transglutaminases.

Transglutaminases (E.C. 2.3.2.13) catalyze the post-translational modification of proteins by establishing ε-(γ-glutamyl) lysine isopeptide bonds and by the covalent conjugation of polyamines to endo-glutamyl residues of proteins. In light of the confirmed role of transglutaminases in animal cell apoptosis and only limited information on the role of these enzymes in plant senescence, we decided to investigate the activity of chloroplast transglutaminases (ChlTGases) and the fate of chloroplast-associated polyamines in Hordeum vulgare L. ‘Nagrad’ leaves, where the senescence process was induced by darkness (day 0) and continued until chloroplast degradation (day 12). Using an anti-TGase antibody, we detected on a subcellular level, the ChlTGases that were associated with destacked/degraded thylakoid membranes, and beginning on day 5, were also found in the stroma. Colorimetric and radiometric assays revealed during senescence an increase in ChlTGases enzymatic activity. The MS/MS identification of plastid proteins conjugated with exogenous polyamines had shown that the ChlTGases are engaged in the post-translational modification of proteins involved in photosystem organization, stress response, and oxidation processes. We also computationally identified the cDNA of Hv-Png1-like, a barley homologue of the Arabidopsis AtPng1 gene. Its mRNA level was raised from days 3 to 10, indicating that transcriptional regulation controls the activity of barley ChlTGases. Together, the presented results deepen our knowledge of the mechanisms of the events happened in dark-induced senescence of barley leaves that might be activation of plastid transglutaminases.

Lead-stress induced changes in the content of free, thylakoid- and chromatin-bound polyamines, photosynthetic parameters and ultrastructure in greening barley leaves

The aim of this study was to determine the impact of lead (Pb) stress as 0.6mM Pb(NO3)2 on the content of free, thylakoid- and chromatin-bound polyamines (PAs) and diamine oxidase (DAO) activity in detached greening barley leaves. Additionally, photosynthetic-related parameters, generation of hydrogen peroxide (H2O2) and malondialdehyde (MDA) content and ultrastructural changes under Pb-stress were studied. The level of putrescine (Put) was reduced progressively to 56% at 24h of Pb stress, and it was correlated with 38% increase of DAO activity. Spermidine (Spd) content was not affected by Pb-stress, while the free spermine (Spm) level significantly increased by about 83% at 6h, and in that time the lowest level of H2O2 was observed. The exogenous applied Spm to Pb-treated leaves caused a decrease in the content of H2O2. In greening leaves exposed to Pb an accumulation of chlorophylls a and b was inhibited by about 39 and 47%, respectively, and photosynthetic parameters of efficiency of electron transport and photochemical reaction in chloroplasts as ΦPSII, ETR and RFd were lowered by about 23-32%. The level of thylakoid-bound Put decreased by about 22%. Moreover, thylakoids isolated from chloroplasts of Pb-treated leaves were characterized with lower Put/Spm ratio as compared to control leaves. In the presence of Pb the significant decrease in the number of thylakoids per granum and cap-shape invaginations of cytoplasmic material were noticed. In Pb-stressed leaves the level of chromatin-bound Spm increased by about 48% and sometimes condensed chromatin in nuclei was observed. We conclude that in greening barley leaves exposed to Pb-stress changes in free, thylakoid- and chromatin-bound PAs play some role in the functioning of leaves or plants in heavy metal stress conditions.

Role of polyamines in the cytokinin-dependent physiological processes. I. Effect of benzyladenine on polyamine levels during chloroplast differentiation in the tissue culture of Dianthus caryophyllus

Tissue culture of Dianthus caryophyllus L. (cv. William Sim.) obligatory requiring N6-benzyladenine for greening provides a good system to study the interactions between cytokinins and polyamines. Polyamines were analyzed as dansyl derivatives which are separated by thin layer chromatography and detected by fluorescence spectrophotometry.Green callus growing on benzyladenine — containing medium showed decrease in the contents of free, conjugated and bound putrescine and spermidine in comparison to chlorophyll-less callus (control callus) growing on cytokinin-free medium. The level of spermine free, conjugated and bound forms increased about 6 %, 77 % and 28 % respectively in tissue culture growing in the presence of cytokinin.Spermidine was dominant polyamine bound to chromatin isolated from control callus. Chromatin isolated from green callus was characterized by a lower level of each polyamine in comparison to chlorophyll-less callus.Polyamines were found in plastid membrane fraction isolated from chlorophyll-less and green callus. A significant increase the levels of polyamines (putrescine, spermidine and spermine) bound to plastid membranes in green callus (+ benzyladenine) in comparison to chlorophyll-less callus (− benzyladenine) was observed. Additionaly, methylglyoxal-bis(guanylhydrazone) an inhibitor of S-adenosylmethionine decarboxylase depressed the greening process.Our results suggest that cytokinin-induced chloroplast differentiation in carnation tissue culture may be partly mediated through the polyamines bound to thylakoid membranes. A possible role of polyamines during cytokinin-induced formation of photosynthetic apparatus is discussed.

Role of polyamines in cytokinin-dependent physiological processes. III. Changes in polyamine levels during cytokinin-induced formation of gametophore buds in Ceratodon purpureus

Cytokinin-induced gametophore bud formation in the protonema of Ceratodon purpureus was used as a model system to study a possible relationship between cytokinin and polyamines. The levels of free, conjugated and bound to chromatin and ribosomes putrescine, spermidine and spermine in moss growing in a medium with or without kinetin was investigated. Cytokinin elevated (about 2-fold) the level of free, conjugated and bound to the PCA-insoluble fraction putrescine and it was correlated with a 2-fold increase in arginine decarboxylase activity. A higher level of spermine bound to chromatin and spermidine bound to ribosomes in cytokinin-treated protonema can play an important role in cytokinin-evoked stimulation of transcription and translation processes (Gwóźdź and Szweykowska 1982, Schneider et al. 1988). This suggests that polyamines may be involved in cytokinin mode of action during the induction of gametophore bud formation.This suggestion was additionaly supported by the experiments with inhibitors of putrescine synthesis, D-arginine and canavanine as well as spermidine synthesis, MGBG, which when added to the medium together with kinetin, reduced or inhibited cytokinin-stimulated gametophore bud formation.

From Accumulation to Degradation: Reprogramming Polyamine Metabolism Facilitates Dark-Induced Senescence in Barley Leaf Cells

The aim of this study was to analyze whether polyamine (PA) metabolism is involved in dark-induced Hordeum vulgare L. ‘Nagrad’ leaf senescence. In the cell, the titer of PAs is relatively constant and is carefully controlled. Senescence-dependent increases in the titer of the free PAs putrescine, spermidine, and spermine occurred when the process was induced, accompanied by the formation of putrescine conjugates. The addition of the anti-senescing agent cytokinin, which delays senescence, to dark-incubated leaves slowed the senescence-dependent PA accumulation. A feature of the senescence process was initial accumulation of PAs at the beginning of the process and their subsequent decrease during the later stages. Indeed, the process was accompanied by both enhanced expression of PA biosynthesis and catabolism genes and an increase in the activity of enzymes involved in the two metabolic pathways. To confirm whether the capacity of the plant to control senescence might be linked to PA, chlorophyll fluorescence parameters, and leaf nitrogen status in senescing barley leaves were measured after PA catabolism inhibition and exogenously applied γ-aminobutyric acid (GABA). The results obtained by blocking putrescine oxidation showed that the senescence process was accelerated. However, when the inhibitor was applied together with GABA, senescence continued without disruption. On the other hand, inhibition of spermidine and spermine oxidation delayed the process. It could be concluded that in dark-induced leaf senescence, the initial accumulation of PAs leads to facilitating their catabolism. Putrescine supports senescence through GABA production and spermidine/spermine supports senescence-dependent degradation processes, is verified by H2O2 generation.

Kinetin-induced Changes in the Population of Translable Messenger RNA in Cucumber Cotyledons

Summary The poly(A)RNA fraction from cucumber cotyledons (Cucumis sativus L. cv. Monastyrski) treated in darkness with kinetin for 5 and 24 h was isolated and submitted to translation in wheat germ cell-free system. Translational activity of poly(A)RNA after kinetin treatment increased by 40 % as compared with the control. For a comparison, the in vitro made polypeptides were fractionated by two-dimensional polyacrylamide gel electrophoresis. The analysis revealed the presence of a single polypeptide of a mol. wt. 20 kd among translation products of poly(A)RNA from kinetintreated tissue that was absent among polypeptides coded by the control mRNA. Moreover, quantitative changes of certain polypeptides were observed in the kinetin variant. It is postulated that cytokinins are involved in the synthesis of a few sets of proteins at the transcription.al level. Expression of certain mRNA species is modulated at early phases of cytokinin stimulation during the growth of cucumber cotyledons.

The Role of Cytokinins in the Development and Metabolism of Barley Leaves: V. Effect of Light and Kinetin on the Transcriptional Activity of Chromatin in Etiolated Leaves

Summary During the first six hours after illumination of etiolated barley leaves the transcriptional activity of chromatin rapidly increased. The intensity of this light-induced reaction was greatly influenced by leaf age. Kinetin treatment affected chromatin activity, but the response differed also with leaf age. In younger leaves (7-days old) kinetin treatment diminished the effect of light, while in older leaves (9 to l0-days old) kinetin additionally stimulated the activity of chromatin. The observed responses to light and kinetin were primarily due to the α-amanitin-resistant activity, so it is concluded that they were connected with the transcription controlled by RNA polymerase I responsible for the rRNA synthesis. These findings are discussed in relation to the greening process of etiolated leaves and involvement of cytokinins in this process.