Polyamine-Induced Hormonal Changes in eds5 and sid2 Mutant Arabidopsis Plants.

Polyamines are multifaceted compounds which play a role in regulating plant growth and stress tolerance in interactions with plant hormones. The aim of the present study was to reveal how exogenous polyamines influence the synthesis of salicylic acid, with a special emphasis on the effect of salicylic acid deficiency on the polyamine metabolism and polyamine-induced changes in other plant hormone contents. Our hypothesis was that the individual polyamines induced different changes in the polyamine and salicylic acid metabolism of the wild type and salicylic acid-deficient Arabidopsis mutants, which in turn influenced other hormones. To our knowledge, such a side-by-side comparison of the influence of eds5-1 and sid2-2 mutations on polyamines has not been reported yet. To achieve our goals, wild and mutant genotypes were tested after putrescine, spermidine or spermine treatments. Polyamine and plant hormone metabolism was investigated at metabolite and gene expression levels. Individual polyamines induced different changes in the Arabidopsis plants, and the responses were also genotype-dependent. Polyamines upregulated the polyamine synthesis and catabolism, and remarkable changes in hormone synthesis were found especially after spermidine or spermine treatments. The sid2-2 mutant showed pronounced differences compared to Col-0. Interactions between plant hormones may also be responsible for the observed differences.

Corrigendum: Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits.

[This corrects the article on p. 191 in vol. 9, PMID: 29520285.].

Chloroplast Glutamine Synthetase, the Key Regulator of Nitrogen Metabolism in Wheat, Performs Its Role by Fine Regulation of Enzyme Activity via Negative Cooperativity of Its Subunits.

Glutamine synthetase (GS) is of central interest as the main route of ammonia assimilation in plants, and as a connection point between the organic and inorganic worlds. Even though GS activity is critical for producing high yields of crop plants, the autoregulation of substrate consumption of wheat GS remained unknown until now. Here we show kinetic evidence, that the chloroplast localized GS isoform (GS2) of wheat (Triticum aestivum L. cv. Jubilejnaja-50) takes place at the carbon-nitrogen metabolic branch point, where it is a mediator, and its enzymatic activity is regulated in a negatively cooperative allosteric manner. We have discovered that GS2 activity is described by a tetraphasic kinetic curve in response to increasing levels of glutamate supply. We constructed a model that explains the kinetic properties of glutamate consumption and this unique allosteric behavior. We also studied the subunit composition of both wheat leaf GS isoenzymes by a combination of two dimensional gel electrophoresis and protein blotting. Both leaf isozymes have homogeneous subunit composition. Glutamate is both a substrate, and an allosteric regulator of the biosynthetic reaction. We have concluded on the basis of our results and previous reports, that wheat GS2 is probably a homooctamer, and that it processes its substrate in a well-regulated, concentration dependent way, as a result of its negatively cooperative, allosteric activity. Thus, GS2 has a central role as a regulator between the nitrogen and the carbon cycles via maintaining glutamine-glutamate pool in the chloroplast on the level of substrates, in addition to its function in ammonia assimilation.

The effects of putrescine are partly overlapping with osmotic stress processes in wheat.

Polyamine metabolism is in relation with several metabolic pathways and linked with plant hormones or signalling molecules; in addition polyamines may modulate the up- or down-regulation of gene expression. However the precise mechanism by which polyamines act at the transcription level is still unclear. In the present study the modifying effect of putrescine pre-treatment has been investigated using the microarray transcriptome profile analysis under the conditions where exogenous putrescine alleviated osmotic stress in wheat plants. Pre-treatment with putrescine induced the unique expression of various general stress-related genes. Although there were obvious differences between the effects of putrescine and polyethylene glycol treatments, there was also a remarkable overlap between the effects of putrescine and osmotic stress responses in wheat plants, suggesting that putrescine has already induced acclimation processes under control conditions. The fatty acid composition in certain lipid fractions and the antioxidant enzyme activities have also been specifically changed under osmotic stress conditions or after treatment with putrescine.

Polyamines may influence phytochelatin synthesis during Cd stress in rice.

Although the metabolism of phytochelatins and higher polyamines are linked with each other, the direct relationship between them under heavy metal stress has not yet been clarified. Two approaches were used to reveal the influence of polyamine content on cadmium stress responses, particularly with regard to phytochelatin synthesis: putrescine pre-treatment of rice plants followed by cadmium stress, and treatment with the putrescine synthesis inhibitor, 2-(difluoromethyl)ornithine combined with cadmium treatment. The results indicated that putrescine pre-treatment enhanced the adverse effect of cadmium, while the application of 2-(difluoromethyl)ornithine reduced it to a certain extent. These differences were associated with increased polyamine content, more intensive polyamine metabolism, but decreased thiol and phytochelatin contents. The gene expression level and enzyme activity of phytochelatin synthase also decreased in rice treated with putrescine prior to cadmium stress, compared to cadmium treatment alone. In contrast, the inhibition of putrescine synthesis during cadmium treatment resulted in higher gene expression level of phytochelatin synthase. The results suggest that polyamines may have a substantial influence on phytochelatin synthesis at several levels under cadmium stress in rice.

Arabidopsis RETINOBLASTOMA RELATED directly regulates DNA damage responses through functions beyond cell cycle control.

The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Physiological and molecular responses to heavy metal stresses suggest different detoxification mechanism of Populus deltoides and P. x canadensis.

Plants have divergent defense mechanisms against the harmful effects of heavy metals present in excess in soils and groundwaters. Poplars (Populus spp.) are widely cultivated because of their rapid growth and high biomass production, and members of the genus are increasingly used as experimental model organisms of trees and for phytoremediation purposes. Our aim was to investigate the copper and zinc stress responses of three outstanding biomass producer bred poplar lines to identify such transcripts of genes involved in the detoxification mechanisms, which can play an important role in the protection against heavy metals. Poplar cuttings were grown hydroponically and subjected to short-term (one week) mild and sublethal copper and zinc stresses. We evaluated the effects of the applied heavy metals and the responses of plants by detecting the changes of multiple physiological and biochemical parameters. The most severe cellular oxidative damage was caused by 30µM copper treatment, while zinc was less harmful. Analysis of stress-related transcripts revealed genotype-specific differences that are likely related to alterations in heavy metal tolerance. P. deltoides clones B-229 and PE 19/66 clones were clearly more effective at inducing the expression of various genes implicated in the detoxification process, such as the glutathione transferases, metallothioneins, ABC transporters, (namely PtGSTU51, PxMT1, PdABCC2,3), while the P. canadensis line M-1 accumulated more metal, resulting in greater cellular oxidative damage. Our results show that all three poplar clones are efficient in stress acclimatization, but with different molecular bases.

Metabolic indicators of drought stress tolerance in wheat: glutamine synthetase isoenzymes and Rubisco.

Drought stress has a considerable impact on the ecosystem and agriculture. Continuous water deficit induces early leaf senescence in plants. During this process, chloroplasts are degraded and photosynthesis drastically drops. The objective of this investigation was to look into the regulation of nitrogen and carbon metabolism during water deficit. Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) and the total protein contents inform us of the sink-source relation in plants. Glutamine synthetase (GS, EC 6.3.1.2) isoenzymes are good markers of plastid status (GS2) and the nitrogen metabolism (GS1). Tolerant and sensitive wheat (Triticum aestivum L.) genotypes were tested, which are widely used in agriculture. The amount of protein, Rubisco and GS isoforms in leaves were measured during the grain filling period, as indicative traits that ultimately determine the onset and stage of senescence. The symptoms of senescence first appeared on the oldest and finally on the youngest leaves. Drought stress disrupted the sequentiality of senescence in the sensitive varieties. An untimely senescence appeared in flag leaves, earlier than in the older leaves. Total protein and Rubisco contents decreased and the GS2 isoenzyme declined considerably in the youngest leaves. In the tolerant varieties, however, these physiological parameters did not change under drought, only the sequential senescence of leaf levels accelerated in some cases compared to the control, well-watered plants. Our results revealed that GS is a good indicator of drought stress, which can be applied for the characterization of wheat cultivars in terms of drought stress tolerance.

Effect of nursing methods and faeces consumption on the development of the bacteroides, lactobacillus and coliform flora in the caecum of the newborn rabbits.

The effect of nursing method and ingestion of maternal faeces on the development of the bacteroides, lactobacillus and coliform flora of the caecum in the first 10 days of life were examined in freely nursed pups having access to maternal faeces (Group FF), pups nursed once a day and having access (Group CF), or having no access (Group CN) to maternal faeces. Colonisation of the caecum by Bacteroides commenced already on day 3 after birth. On day 2 the bacteroides counts were below 100, while on day 4 they were already between 100 and 10,000. In Group CN, the Bacteroides counts were lower (by 14 to 40%) throughout the 10-day period studied than in the groups having access to maternal faeces. Differences between groups were significant only on days 4 and 6. The average number of maternal faecal pellets left behind the doe in Group CN was 3-4 (between 0.5 and 6.4 per doe). In Groups FF and CF the pellets became smaller, crumbled and finally disappeared from the nest box, they were consumed by the pups and could be found in their gastric content. The lactobacillus counts decreased in all three groups with age, from 6.0 to 3.5 log10 CFU.g-1 (FF), 4.6 to 2.8 log10 CFU.g-1 (CF) and 5.1 to 3.1 log10 CFU.g-1 (CN), respectively. The coliform counts were higher in the first 4 days in FF (5.6 log10 CFU.g-1) than in CF (< 2 log10 CFU.g-1) and CN (2-3.6 log10 CFU.g-1) animals. Bacteroides could be cultured from the surface of the vulvar labia (max. 1000 colony count) and the vagina (max. 190 colony count), so young rabbits could become "infected" by them already in the doe's vagina. Thus prevention of ingestion of maternal faeces only slightly influenced the development of the bacteroides flora, the faeces left behind by the doe did not play an exclusive role in their colonisation.