No abstract

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.

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.List of abbreviations." Chl -chlorophyll; LHCIIlight harvesting chlorophyll a/b-protein complex; LHCPII -apoprotein of the light-harvesting chlorophyll a/b-protein complex of photosystem II; PAspolyamines; PCA -perchloric acid; PSII -photosystem II; Put -putrescine; Spd -spermidine; Spmspermine; SDS-PAGE -sodium dodecylsulphate polyacrylamide gel electrophoresis.

We analysed the level of polyamines (PAs) bound to thylakoids and the level and activity of thylakoid transglutaminases throughout barley leaf senescence, retarded by kinetin. The level of PAs bound to thylakoids changed in senescing barley leaves: bound putrescine (PU) and spermidine (SD) increased throughout senescence, whereas bound spermine (SM) decreased. Kinetin diminished the increase in thylakoid-bound PU and SD and almost completely abolished the decrease of the bound SM. These data suggest different roles of PU/SD and SM in thylakoid degradation. Immunodetection of transglutaminases (TGase) in thylakoid fraction revealed three bands of 33, 58 and 78 kDa. During senescence the intensity of all bands increased and it was correlated with an increase in TGase activity. Kinetin down-regulated the accumulation of the 58- and 78-kDa TGases and the TGase activity. We postulate that formation of covalent bonds between PAs and proteins by TGase is involved in chloroplast senescence. The kinetin-mediated preservation of low TGase levels and activity throughout leaf senescence may represent an important component of the mechanism of kinetin action in the retardation of leaf senescence.

We have examined the effects of cytokinin, fusicoccin, and ethylene on auxin-induced changes in gene expression during auxin-promoted cell elongation in soybean (Glycine max L. Meff. cv Wayne) using cloned cDNAs to two auxin-responsive mRNAs (Walker, Key 1982 Proc Natl Acad Sci USA 79: . RNA blot analyses demonstrate that under conditions of cytokinin inhibition of auxin-promoted cell elongation the levels of these two auxin-responsive mRNAs is unaltered. Fusicoccinpromoted elonption is not associated with an enhanced expression of these two mRNAs, suggesting that the increased levels of these mRNAs observed during auxin-promoted cell elongation are not simply due to enhanced rates of cell elonption. We have also determined that ethylene plays no apparent role in the regulation of expression of these mRNAs. However, the auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and a-naphthalene acetic acid all enhance an accumulation of these mRNAs. We conclude that the regulation of these mRNAs is directly dependent on auxin. That auxin-promoted cell elongation is dependent upon the increased accumulation of these mRNAs remains to be determined.In recent years a considerable amount of evidence has accumulated indicating that all classes of plant hormones can cause selective changes in the levels of specific mRNAs (e.g. 3, 6, 8, 21). One system that has proven to be a useful model for studying the molecular mechanisms underlying hormonally induced changes in gene expression has been auxin-induced cell elongation (e.g. 12, 18, 23, 26 23, 25, and 50 uM, respectively (see 11 and references cited therein); FC was used at 10 JuM (see 14 and references cited therein); IPA was used at 50 uM (22). In experiments using ethylene, ethylene in air (10 ul/1) was gently bubbled into the incubation medium and allowed to exit through a second hole in the stoppered flask into 50% (v/v) ethanol. AVG was used at 100 ,M and ACC at 1 mM (9); all incubations using these compounds were performed in stoppered flasks. IAA, 2,4-D, a-NAA, IPA, AOA, and ACC were obtained from Sigma; AVG was a gift of W

The amounts of polyamines (PAs) bound to etioplast membranes varied during chloroplast development in cucumber cotyledons (Cucumis sativus L. cv. Racibór). Putrescine (PU) and spermidine (SD) levels increased in the early greening stage (6 h of light exposure) but decreased in the late greening stage (24 h) in the thylakoid‐enriched fraction. In the highly enriched PSIIα fraction, the trend of changes in the amount of bound PAs was different: levels of SD and spermine (SM) increased in the late stage. In both fractions, their levels were additionally increased by kinetin treatment. In the presence of exogenous protein transglutaminase (TGase) substrate (N′,N′‐dimethylcasein) and 5 mM Ca2+, kinetin initially caused a marked increase in thylakoid transglutaminase (ThylTGase) activity (6 h), followed by a decrease at the end of greening. The radiometric assay showed that PU and SM binding to thylakoid proteins was very low, while SD binding was seven to eight times higher. Kinetin increased SD conjugation in the early greening stage by about 36%. When chloroplast membranes were fully organized, ThylTGase activity decreased. In etioplast membranes and during the early greening stage, the 77‐kDa and 30‐kDa bands were mainly immunodetected with antibodies raised against the animal TGase, which were in general slightly stronger for kinetin‐treated than the control samples. At the end of greening, the level of 77‐kDa ThylTGase dramatically decreased. ThylTGase activity was found to be Ca2+ dependent. PAs conjugated via ThylTGase, in addition to the PAs bound by all possible types of linkage, could represent an important component of the mechanism of stimulation of etioplast‐to‐chloroplast transformation by kinetin.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1912-y) contains supplementary material, which is available to authorized users.

Polyamines are low-molecular weight biogenic amines. They are a specific group of cell growth and development regulators. In the past decade biochemical, molecular and genetic studies have contributed much to a better understanding of the biological role of polyamines in the plant cell. Substantial evidence has also been added to our understanding of the role of polyamines in plastid development. In developing chloroplasts, polyamines serve as a nitrogen source for protein and chlorophyll synthesis. In chloroplast structure, thylakoid proteins linked to polyamines belong mainly to antenna proteins of light-harvesting chlorophyll a/b-protein complexes. The fact that LHCII oligomeric forms are much more intensely labelled by polyamines, in comparison to monomeric forms, suggests that polyamines participate in oligomer stabilisation. In plastid metabolism, polyamines modulate effectiveness of photosynthesis. The role of polyamines in mature chloroplasts is also related to the photo-adaptation of the photosynthetic apparatus to low and high light intensity and its response to environmental stress. The occurrence of polyamines and enzymes participating in their metabolism at every stage of plastid development indicates that polyamines play a role in plastid differentiation, structure, functioning and senescence.