Search

We explored the polyamine (PA) metabolic pathway genes in barley (Hv) to understand plant development and stress adaptation in Gramineae crops with emphasis on leaf senescence. Bioinformatics and functional genomics tools were utilized for genome-wide identification, comprehensive gene features, evolution, development and stress effects on the expression of the polyamine metabolic pathway gene families (PMGs). Three S-adenosylmethionine decarboxylases (HvSAMDCs), two ornithine decarboxylase, one arginine decarboxylase, one spermidine synthase (HvSPDS), two spermine synthases (HvSPMSs), five copper amine oxidases (HvCuAO) and seven polyamine oxidases (HvPAOs) members of PMGs were identified and characterized in barley. All the genes except for HvCuAO3 were found distributed on all chromosomes of barley. The phylogenetic and comparative assessment revealed that PA metabolic pathway is highly conserved in plants and the prediction of nine H. vulgare miRNAs (hvu-miR) target sites, 18 protein-protein interactions and 961 putative CREs in the promoter region were discerned. Gene expression of HvSAMDC3, HvCuAO7, HvPAO4 and HvSPMS1 were apparent at every developmental stage. SPDS/SPMS gene family was found to be the most responsive to induced leaf senescence. This study provides a reference for the functional investigation of the molecular mechanism(s) that regulate polyamine in plants as a tool for future breeding decision management systems.

Nitric oxide (NO) is a critical molecule that links plant development with stress responses. Herein, new insights into the role of NO metabolism during leaf senescence in Arabidopsis are presented. A gradual decrease in NO emission accompanied dark-induced leaf senescence (DILS), and a transient wave of peroxynitrite (ONOO–) formation was detected by day 3 of DILS. The boosted ONOO– did not promote tryptophan (Trp) nitration, while the pool of 6-nitroTrp-containing proteins was depleted as senescence progressed. Immunoprecipitation combined with mass spectrometry was used to identify 63 and 4 characteristic 6-nitroTrp-containing proteins in control and individually darkened leaves, respectively. The potential in vivo targets of Trp nitration were mainly related to protein biosynthesis and carbohydrate metabolism. In contrast, nitration of tyrosine-containing proteins was intensified 2-fold on day 3 of DILS. Also, nitrative modification of RNA and DNA increased significantly on days 3 and 7 of DILS, respectively. Taken together, ONOO– can be considered a novel pro-senescence regulator that fine-tunes the redox environment for selective bio-target nitration. Thus, DILS-triggered nitrative changes at RNA and protein levels promote developmental shifts during the plant’s lifespan and temporal adjustment in plant metabolism under suboptimal environmental conditions.

Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein–protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.

This review synthesizes knowledge on epigenetic regulation of leaf senescence and discusses the possibility of using this knowledge to improve crop quality. This control level is implemented by different but interacting epigenetic mechanisms, including DNA methylation, covalent histone modifications, and non-covalent chromatin remodeling. The genetic and epigenetic changes may act alone or together and regulate the gene expression, which may result in heritable (stress memory) changes and may lead to crop survival. In the review, the question also arises whether the mitotically stable epigenetic information can be used for crop improvement. The barley crop model for early and late events of dark-induced leaf senescence (DILS), where the point of no return was defined, revealed differences in DNA and RNA modifications active in DILS compared to developmental leaf senescence. This suggests the possibility of a yet-to-be-discovered epigenetic-based switch between cell survival and cell death. Conclusions from the analyzed research contributed to the hypothesis that chromatin-remodeling mechanisms play a role in the control of induced leaf senescence. Understanding this mechanism in crops might provide a tool for further exploitation toward sustainable agriculture: so-called epibreeding.

Barley crop model was analyzed for early and late events during the dark-induced leaf senescence (DILS) as well as for deciphering critical time limit for reversal of the senescence process. Chlorophyll fluorescence vitality index Rfd was determined as the earliest parameter that correlated well with the cessation of photosynthesis prior to microautophagy symptoms, initiation of DNA degradation, and severalfold increase in the endonuclease BNUC1. DILS was found characterized by up-regulation of processes that enable recycling of degraded macromolecules and metabolites, including increased NH 4 + remobilization, gluconeogenesis, glycolysis, and partial up-regulation of glyoxylate and tricarboxylate acid cycles. The most evident differences in gene medleys between DILS and developmental senescence included hormone-activated signaling pathways, lipid catabolic processes, carbohydrate metabolic processes, low-affinity ammonia remobilization, and RNA methylation. The mega-autophagy symptoms were apparent much later, specifically on day 10 of DILS, when disruption of organelles—nucleus and mitochondria —became evident. Also, during this latter-stage programmed cell death processes, namely, shrinking of the protoplast, tonoplast interruption, and vacuole breakdown, chromatin condensation, more DNA fragmentation, and disintegration of the cell membrane were prominent. Reversal of DILS by re-exposure of the plants from dark to light was possible until but not later than day 7 of dark exposure and was accompanied by regained photosynthesis, increase in chlorophyll, and reversal of Rfd, despite activation of macro-autophagy-related genes.

Senescence is the final stage of plant ontogeny before death. Senescence may occur naturally because of age or may be induced by various endogenous and exogenous factors. Despite its destructive character, senescence is a precisely controlled process that follows a well-defined order. It is often inseparable from programmed cell death (PCD), and a correlation between these processes has been confirmed during the senescence of leaves and petals. Despite suggestions that senescence and PCD are two separate processes, with PCD occurring after senescence, cell death responsible for senescence is accompanied by numerous changes at the cytological, physiological and molecular levels, similar to other types of PCD. Independent of the plant organ analysed, these changes are focused on initiating the processes of cellular structural degradation via fluctuations in phytohormone levels and the activation of specific genes. Cellular structural degradation is genetically programmed and dependent on autophagy. Phytohormones/plant regulators are heavily involved in regulating the senescence of plant organs and can either promote [ethylene, abscisic acid (ABA), jasmonic acid (JA), and polyamines (PAs)] or inhibit [cytokinins (CKs)] this process. Auxins and carbohydrates have been assigned a dual role in the regulation of senescence, and can both inhibit and stimulate the senescence process. In this review, we introduce the basic pathways that regulate senescence in plants and identify mechanisms involved in controlling senescence in ephemeral plant organs. Moreover, we demonstrate a universal nature of this process in different plant organs; despite this process occurring in organs that have completely different functions, it is very similar. Progress in this area is providing opportunities to revisit how, when and which way senescence is coordinated or decoupled by plant regulators in different organs and will provide a powerful tool for plant physiology research.

Drought stress is one of the most important abiotic stresses causing disturbance of the intracellular water relations and leads to growth inhibition, impaired photosynthesis and ion homeostasis, decreased seed germination rate, enhanced production of reactive oxygen species, and eventually, reduced yields. Severe water deficit causes stress and could be lethal for most of plants, whereas mild water deficit or short periods of drought stress may enhance stress tolerance by inducing hardening. In agricultural practice, a priming process is artificially initiated for the plant to sustain a stress situation. Some of the possible mechanisms involved in a plant's recall of stress episode (e.g., drought) and enhanced response to the stressor in the future include: alterations in the levels of key signaling molecules, proteins or transcription factors; changes in chromatin status, such as histone tail modifications or DNA methylation; and expression of miRNAs involved in posttranscriptional gene silencing.

Polyamines (PAs) are low molecular weight organic cations comprising biogenic amines that play multiple roles in plant growth and senescence. PA metabolism was found to play a central role in metabolic and genetic reprogramming during dark-induced barley leaf senescence (DILS). Robust PA catabolism can impact the rate of senescence progression in plants. We opine that deciphering senescence-dependent polyamine-mediated multidirectional metabolic crosstalks is important to understand regulation and involvement of PAs in plant death and re-mobilization of nutrients during senescence. This will involve optimizing the use of PA biosynthesis inhibitors, robust transgenic approaches to modulate PA biosynthetic and catabolic genes, and developing novel germplasm enriched in pro- and anti-senescence traits to ensure sustained crop productivity. PA-mediated delay of senescence can extend the photosynthesis capacity, thereby increasing grain starch content in malting grains such as barley. On the other hand, accelerating the onset of senescence can lead to increases in mineral and nitrogen content in grains for animal feed. Unraveling the “polyamine metabolic switch” and delineating the roles of PAs in senescence should further our knowledge about autophagy mechanisms involved in plant senescence as well as mammalian systems. It is noteworthy that inhibitors of PA biosynthesis block cell viability in animal model systems (cell tumor lines) to control some cancers, in this instance, proliferative cancer cells were led toward cell death. Likewise, PA conjugates work as signal carriers for slow release of regulatory molecule nitric oxide in the targeted cells. Taken together, these and other outcomes provide examples for developing novel therapeutics for human health wellness as well as developing plant resistance/tolerance to stress stimuli.

Leaf senescence is a terminal step in plant growth and development. Considerable information on processes and signals involved in this process has been obtained, although comparatively little is known about leaf senescence in monocotyledonous plants. In particular, little is known about players involved in leaf senescence imposed by a prolonged dark treatment. New information has now been unveiled on dark-induced leaf senescence in a monocot, barley. A close association has been found between ubiquitous polyamines, reactive oxygen species (ROS), and senescence of barley leaves during prolonged darkness. Although polyamines (putrescine, spermidine, and spermine) are absolutely essential for critical cellular functions, including regulation of nucleic acids and protein synthesis, macromolecular structural integrity, and signalling, a strong link between polyamines and dark-induced leaf senescence has been found using barley plant as a model of monocots. Interestingly, Arabidopsis polyamine back-conversion oxidase mutants deficient in the conversion of spermine to spermidine and/or spermidine to putrescine do not occur and have delayed entry into dark-induced leaf senescence. This review summarizes the recent molecular, physiological, and biochemical evidence implicating concurrently polyamines and ethylene in dark-induced leaf senescence and broadening our knowledge on the mechanistic events involved in this important plant death process.

Leaf senescence is a regulated developmental program, an endogenous process which is also induced by changes in the environmental factors including darkness, nutrient deficiency, pathogens or abiotic stresses. Research into biochemical and molecular processes involved in the senescence phenomenon has revealed interesting phenomenology and added to our understanding of this cellular phenomenon. Leaf senescence is divided generally into three phases: initiation, cellular degradation, and final termination that leads to cell death and leaf fall. Hormones ethylene, abscisic acid (ABA), brassinosteroids, salicylic acid and jasmonates (MeJa) are considered as promoters while cytokinins and gibberellins as inhibitors of leaf senescence. One of the recently recognized regulators of leaf senescence is a group of the biogenic amines, called polyamines (PAs). PAs constitute diamine putrescine, triamine spermidine, and tetra-amine spermine/thermo-spermine. Since the latter are found in all compartments of a plant cell, they may participate in regulating a specific, or series of, function(s) in any cellular compartment. Recent identification of thermo-spermine (T-Spm), first discovered in thermophilic bacteria, as a stress regulator has attracted attention. In this review we provide a window into the role of PAs in induced-senescence phenomenon, with special reference to barley. We discuss subject matter related to the sensing of cellular fluctuations in PA concentrations; cross-talk between PAs and plant hormones in terms of signaling; nexus between PA catabolism, H2O2, GABA and senescence; PA oxidation-derived H2O2 as a signal generated in the chloroplasts; and finally a perspective on a possible nexus between nitrous oxide (NO), PA and H2O2 as a signaling triad that may regulate leaf senescence.

Problems with understanding concepts and mechanisms connected to plant movements have been diagnosed among biology students. Alternative conceptions in understanding these phenomena are marginally studied. The diagnosis was based on a sample survey of university students and their lecturers, which was quantitatively and qualitatively exploratory in nature (via a questionnaire). The research was performed in two stages, before and after the lectures and laboratory on plant movements. We diagnosed eight alternative conceptions before the academic training started. After the classes, most were not been verified, and in addition, 12 new conceptions were diagnosed. Additionally, we report that teachers are not aware of students’ possible misunderstandings. They do not perceive students’ troubles with switching between levels of representations, nor their alternative conceptions. A case of “curse of knowledge” was observed and academic teacher training is recommended. Additionally, the need for metacognition as a crucial element in laboratory activities seems supported by our presented results. Such metacognition refers to students as well as teachers, which leads to the conclusion that teachers should be aware of students’ way of thinking and the development of knowledge in one’s own mind.

Cell senescence occurs as a part of developmental or stress-induced process. It is tightly regulated and involves a sequence of metabolic and structural alterations, eventually leading to cell death. Dark-induced leaf senescence is a useful model for studying senescence-related events. To facilitate the integration of physiological and molecular studies utilizing this model, we generated the microarray data set providing time course gene expression profiles in senescing barley leaves. Here, we describe the detailed procedures and data analysis scheme of our experiment. The entire data set (available at NCBI/GEO database under GSE62539) has been successively explored to find the genes differentially expressed during the senescence process as well as to identify genes with the invariant expression as reliable references for qPCR or ddPCR experiments.

During greening, excised etiolated barley leaves and cucumber cotyledons that were depleted of exogenous Ca2+ by a chelating agent (ethylene glycol-bis (beta aminoethyl ether)-N,N,N`N`-tetraacetic acid, EGTA) showed ∼50% reduced chlorophyll (Chl) accumulation and ∼30% accumulation of apoprotein of the light-harvesting chlorophyll a/b-binding protein complex of photosystem II (LHCPII). The Ca2+ channel blocker lanthanum chloride (LaCl3) applied to cucumber cotyledons reduced LHCPII accumulation more than EGTA did. In both plant mate-rials, cytokinins enhanced chlorophyll accumulation by 50-60% and this effect was completely canceled by EGTA application. Hormones significantly increased LHCPII accumulation but EGTA application reduced that effect in barley leaves by ∼30% and in cucumber cotyledons by ∼80%. A similar effect was observed in LaCl3-treated cotyledons. CaCl2 application boosted chlorophyll accumulation in both plant materials. CaCl2 applied together with cytokinin reduced the hormonal effect on chlorophyll accumulation by ∼38% in barley leaves and 23% in cucumber cotyledons, but almost totally inhibited cytokinin-stimulated LHCPII accumulation. Our results indicate that calcium variously mediates the effect of cytokinin on chlorophyll and LHCPII accumulation. Cytokinin-induced enhancement of chlorophyll accumulation seems totally dependent on the exogenous pool of Ca2+, while Ca2+-dependent and Ca2+-independent pathways are involved in the hormonal effect on LHCPII accumulation. The effect of cytokinin on the increase of light-induced LHCPII accumulation appears to be sensitive to exogenously applied Ca2+, which almost totally blocked the hormonal effect. Our results give indirect evidence that the responses to cytokinin and light act on different events leading to Chl and LHCPII accumulation.

The levels of polyamines (PAs), proline (Pro), and γ-aminobutyric acid (GABA) as well as the activity of diamine oxidase (DAO; EC 1.4.3.6) were studied in the roots of 2-day-old lupine (Lupinus luteus L. ‘Juno’) seedlings treated with 200 mM NaCl for 24 h. The effect of adding 1 mM aminoguanidine (AG), an inhibitor of DAO activity, was also analyzed. It was found that in roots of lupine seedlings growing under salt stress, a negative correlation between Pro accumulation and putrescine (Put) content takes place. Pro level increased in roots by about 160% and, at the same time, Put content decreased by about 60%, as a result of ca. twofold increase of DAO activity. The AG added to the seedlings almost totally inhibited the activity of DAO, increased Put accumulation to control level, decreased Pro content by about 25%, and reduced GABA level by about 22%. Addition of 50 mM GABA to the lupine seedlings growing in the presence of AG and NaCl restored Pro content in roots to its level in NaCl-treated plants. In this research, the clear correlation between Put degradation and GABA and Pro accumulation was shown for the first time in the roots of seedlings growing under salt stress. This could be considered as a short-term response of a plant to high salt concentration. Our findings indicate that during intensive Pro accumulation in roots induced by salt stress, the pool of this amino acid is indirectly supported by GABA production as a result of Put degradation.

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.

University students often struggle to understand the role of water in plant cells. This study attempted to identify student difficulties (including misconceptions) concerning osmosis and plasmolysis and examined to what extent the difficulties could be ameliorated during a plant physiology course., University students often struggle to understand the role of water in plant cells. In particular, osmosis and plasmolysis appear to be challenging topics. This study attempted to identify student difficulties (including misconceptions) concerning osmosis and plasmolysis and examined to what extent the difficulties could be revised during a plant physiology course. A questionnaire was developed to monitor university students’ conceptual knowledge before and after a course, and both qualitative and quantitative data were obtained. The data were analyzed using the constant comparison technique and descriptive statistics. Students were found to come to university with many misconceptions that had accumulated during their education. These misconceptions are extremely difficult to change during the traditional course, which comprises lectures and practical exercises. Students’ misconceptions originate from commonly used sources such as textbooks, which are perceived as being reliable. Effective teaching of water relations in plant cells could include such didactic methods as “questioning the author,” which allow teachers to monitor students’ knowledge and help students acquire a more scientific understanding of key concepts.

This report deals with the effect of kinetin on the greening process, in relation to endogenous free polyamine levels and their metabolism in cucumber cotyledons. The kinetin response on free polyamine levels was found to be accompanied by an increase in free putrescine throughout the greening process. There was no significant difference in spermidine and spermine levels between control (water-treated) and kinetin-treated cotyledons; however, a slight increase in spermidine level, which was higher in control was observed at 4 h. In order to examine the action of kinetin on polyamine metabolism, particularly spermidine synthesis, the effect of kinetin on the level of S-adenosylmethionine decarboxylase mRNA and its enzyme activity were studied. First, an increase in the S-adenosylmethionine decarboxylase transcript level was observed at 30 min of illumination in water and kinetin-treated cotyledons, and next, the transcript decreased and was restored again at 2 h in kinetin-treated cotyledons and at 4 h in the control. This is the first report that demonstrates the light and kinetin regulation of S-adenosylmethionine decarboxylase transcript level. The highest S-adenosylmethionine decarboxylase activity was observed at 2 h of illumination, and it was higher in control when compared to kinetin-treated cotyledons. Spermidine and spermine levels observed in kinetin-treated cotyledons at 4 h of illumination may partly be a result of: lower S-adenosylmethionine decarboxylase activity inhibited by kinetin and/or higher by about 35% on kinetin polyamine oxidase activity. Experiments with methylglyoxal-bis (guanylhydrazone) and dicyclohexylamine showed that both spermidine synthesis inhibitors depressed chlorophyll accumulation in the greening cucumber cotyledons. Additionally, these results, indirectly confirm that polyamines may play some role in the greening process stimulated by kinetin.

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.

Programmed cell death (PCD) is a process that occurs throughout the life span of every plant life, from initial germination of the seed to the senescence of the plant. It is a normal physiological milestone during the plant's developmental process, but it can also be induced by external factors, including a variety of environmental stresses and as a response to pathogen infections. Changes in the morphology of the nucleus is one of the most noticeable during PCD but all the components of the plant cell (cytoplasm, cytoskeleton and organelles) are involved in this fascinating process. To date, relatively little is known about PCD in plants, but several factors, among which polyamines (PAs) and plant growth regulators, have been shown to play an important role in the initiation and regulation of the process. The role of PAs in plant PCD appears to be multifaceted acting in some instances as pro-survival molecules, whereas in others seem to be implicated in accelerating PCD. The molecular mechanism is still under study. Here we present some PCD plant models, focusing on the role of the enzyme responsible for PA conjugation to proteins: transglutaminase (TGase), an enzyme linked with the process of PCD also in some animal models. The role of PAs and plant TGase in the senescence and PCD in flowers, leaf and the self-incompatibility of pollen will be discussed and examined in depth.

Leaf senescence is a tightly regulated developmental or stress-induced process. It is accompanied by dramatic changes in cell metabolism and structure, eventually leading to the disintegration of chloroplasts, the breakdown of leaf proteins, internucleosomal fragmentation of nuclear DNA and ultimately cell death. In light of the global and intense reorganization of the senescing leaf transcriptome, measuring time-course gene expression patterns in this model is challenging due to the evident problems associated with selecting stable reference genes. We have used oligonucleotide microarray data to identify 181 genes with stable expression in the course of dark-induced senescence of barley leaf. From those genes, we selected 5 candidates and confirmed their invariant expression by both reverse transcription quantitative PCR and droplet digital PCR (ddPCR). We used the selected reference genes to normalize the level of the expression of the following senescence-responsive genes in ddPCR assays: SAG12, ICL, AGXT, CS and RbcS. We were thereby able to achieve a substantial reduction in the data variability. Although the use of reference genes is not considered mandatory in ddPCR assays, our results show that it is advisable in special cases, specifically those that involve the following conditions: i) a low number of repeats, ii) the detection of low-fold changes in gene expression or iii) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.

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

In the light of our previous work, we know that there is a relationship between bound polyamines and the chloroplast differentiation process. This relationship may represent an important component of the process and be part of the mechanism of kinetin action, which stimulates chloroplast differentiation. To clarify the nature of the binding of polyamines to chloroplast structures, the possible involvement of transglutaminases in kinetin-stimulated chloroplast photodevelopment was investigated. Immunodetection of transglutaminases revealed bands at 77, 50 and 30 kDa both in etioplasts and chloroplasts. The data indicated a positive correlation between enzyme level and activity. It also demonstrated the regulation of transglutaminase protein expression by kinetin. The suborganellar location of transglutaminases by electron microscopy showed that the enzyme is peculiarly localised, mainly in pro-thylakoids and appressed grana thylakoids. The data corroborated that spermidine post-translational modification of certain plastid proteins of 58, 29, 26 and 12 kDa occurred. The results we obtained suggest that transglutaminases take part in the formation of the chloroplast structure via a mechanism whereby polyamines bind to their protein substrates. These findings about the effect of kinetin on conjugation provide a new contribution to the understanding of the mechanism of kinetin action on etioplast-to chloroplast transformation.

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 5mM 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 5 to 7 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.

Most of commercially important crops, including maize and common bean, are sensitive to water deficit and salinity. Polyamines are considered to be osmotic and salt tolerance modulators and biochemical indicators of these stresses. In the present study, we measured organ-specific changes in levels of free, microsome- and thylakoid-associated polyamines in leaves and roots of maize and common bean plants exposed for 24 h to osmotic and saline stresses. Putrescine levels were generally higher in the studied organs of both species and under both stresses; only in the roots of salt-treated bean it considerably decreased. In both species, salt stress (200 mM NaCl) induced a significant decrease in free spermidine in roots. We observed a significant decrease in the contents of all polyamines associated with the microsomes isolated from the roots of maize and bean growing in sorbitol and salt conditions. Also the microsomes isolated from the leaves of stressed plants were characterized by the lower contents of polyamines. Our data showed a reduction of putrescine content, with significantly decreased spermidine and spermine levels in thylakoids isolated from the chloroplasts of maize and bean plants growing under both stresses. The results indicate that the studied maize and bean cultivars are rather drought-sensitive. Additionally, microsome- and thylakoid-associated polyamines seem to be good markers of plant stress tolerance.

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.