Leaf yellowing and anthocyanin accumulation are two genetically independent strategies in response to nitrogen limitation in Arabidopsis thaliana

For the first time in Arabidopsis thaliana, this work proposes the identification of quantitative trait loci (QTLs) associated with leaf senescence and stress response symptoms such as yellowing and anthocyanin-associated redness. When Arabidopsis plants were cultivated under low nitrogen conditions, we observed that both yellowing of the old leaves of the rosette and whole rosette redness were promoted. Leaf yellowing is a senescence symptom related to chlorophyll breakdown. Redness is a symptom of anthocyanin accumulation related to whole plant ageing and nutrient limitation. In this work, Arabidopsis is used as a model system to dissect the genetic variation of these parameters by QTL mapping in the 415 recombinant inbred lines of the Bay-0xShahdara population. Fifteen new QTLs and two epistatic interactions were described in this study. The yellowing of the rosette, estimated by visual notation and image processing, was controlled by four and five QTLs, respectively. The visual estimation of redness allowed us to detect six QTLs among which the major one explained 33% of the total variation. Two main QTLs were confirmed in near-isogenic lines (heterogenous inbred family; HIF), thus confirming the relevance of the visual notation of these traits. Co-localizations between QTLs for leaf yellowing, redness and nitrogen use efficiency described in a previous publication indicate complex interconnected pathways involved in both nitrogen management and senescence- and stress-related processes. No co-localization between QTLs for leaf yellowing and redness has been found, suggesting that the two characters are genetically independent.     

The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8‐Br‐cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen‐activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non‐functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.     

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Effects of heterocyclic and tertiary permeant amines on the electron transfer in thylakoid membranes

The effect of low concentrations (up to 50 μM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H₂O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5–8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10–15% inhibition of transport. With all the amines, the rate of electron transport from H₂O to DCBQ, PS II acceptor is decreased to 80–90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7–6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction. This revised version was published online in June 2006 with corrections to the Cover Date.     

Classical anticytokinins do not interact with cytokinin receptors but inhibit cyclin-dependent kinases

Cytokinins are a class of plant hormones that regulate the cell cycle and diverse developmental and physiological processes. Several compounds have been identified that antagonize the effects of cytokinins. Based on structural similarities and competitive inhibition, it has been assumed that these anticytokinins act through a common cellular target, namely the cytokinin receptor. Here, we examined directly the possibility that various representative classical anticytokinins inhibit the Arabidopsis cytokinin receptors CRE1/AHK4 (cytokinin response 1/Arabidopsis histidine kinase 4) and AHK3 (Arabidopsis histidine kinase 3). We show that pyrrolo[2,3-d]pyrimidine and pyrazolo[4,3-d]pyrimidine anticytokinins do not act as competitors of cytokinins at the receptor level. Flow cytometry and microscopic analyses revealed that anticytokinins inhibit the cell cycle and cause disorganization of the microtubular cytoskeleton and apoptosis. This is consistent with the hypothesis that they inhibit regulatory cyclin-dependent kinase (CDK) enzymes. Biochemical studies demonstrated inhibition by selected anti-cytokinins of both Arabidopsis and human CDKs. X-ray determination of the crystal structure of a human CDK2-anticytokinin complex demonstrated that the antagonist occupies the ATP-binding site of CDK2. Finally, treatment of human cancer cell lines with anticytokinins demonstrated their ability to kill human cells with similar effectiveness as known CDK inhibitors.     

A chemically induced new pea (Pisum sativum) mutant SGECdt with increased tolerance to, and accumulation of, cadmium

BACKGROUND AND AIMS: To date, there are no crop mutants described in the literature that display both Cd accumulation and tolerance. In the present study a unique pea (Pisum sativum) mutant SGECd(t) with increased Cd tolerance and accumulation was isolated and characterized. METHODS: Ethylmethane sulfonate mutagenesis of the pea line SGE was used to obtain the mutant. Screening for Cd-tolerant seedlings in the M2 generation was performed using hydroponics in the presence of 6 microm CdCl2. Hybridological analysis was used to identify the inheritance of the mutant phenotype. Several physiological and biochemical characteristics of SGECd(t) were studied in hydroponic experiments in the presence of 3 microm CdCl2, and elemental analysis was conducted. KEY RESULTS: The mutant SGECd(t) was characterized as having a monogenic inheritance and a recessive phenotype. It showed increased Cd concentrations in roots and shoots but no obvious morphological defects, demonstrating its capability to cope well with increased Cd levels in its tissues. The enhanced Cd accumulation in the mutant was accompanied by maintenance of homeostasis of shoot Ca, Mg, Zn and Mn contents, and root Ca and Mg contents. Through the application of La(+3) and the exclusion of Ca from the nutrient solution, maintenance of nutrient homeostasis in Cd-stressed SGECd(t) was shown to contribute to the increased Cd tolerance. Control plants of the mutant (i.e. no Cd treatment) had elevated concentrations of glutathione (GSH) in the roots. Through measurements of chitinase and guaiacol-dependent peroxidase activities, as well as proline and non-protein thiol (NPT) levels, it was shown that there were lower levels of Cd stress both in roots and shoots of SGECd(t). Accumulation of phytochelatins [(PCcalculated) = (NPT)-(GSH)] could be excluded as a cause of the increased Cd tolerance in the mutant. CONCLUSIONS: The SGECd(t) mutant represents a novel and unique model to study adaptation of plants to toxic heavy metal concentrations.     

Efficient assembly of photosystem II in Chlamydomonas reinhardtii requires Alb3.1p, a homolog of Arabidopsis ALBINO3

Alb3 homologs Oxa1 and YidC have been shown to be required for the integration of newly synthesized proteins into membranes. Here, we show that although Alb3.1p is not required for integration of the plastid-encoded photosystem II core subunit D1 into the thylakoid membrane of Chlamydomonas reinhardtii, the insertion of D1 into functional photosystem II complexes is retarded in the Alb3.1 deletion mutant ac29. Alb3.1p is associated with D1 upon its insertion into the membrane, indicating that Alb3.1p is essential for the efficient assembly of photosystem II. Furthermore, levels of nucleus-encoded light-harvesting proteins are vastly reduced in ac29; however, the remaining antenna systems are still connected to photosystem II reaction centers. Thus, Alb3.1p has a dual function and is required for the accumulation of both nucleus- and plastid-encoded protein subunits in photosynthetic complexes of C. reinhardtii.     

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 μM), sodium nitroprusside (5 μM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1′-1′ Diphenyl-2′ picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.     

Mitochondria as ATP consumers in cellular pathology

ATP provided by oxidative phosphorylation supports highly complex and energetically expensive cellular processes. Yet, in several pathological settings, mitochondria could revert to ATP consumption, aggravating an existing cellular pathology. Here we review (i) the pathological conditions leading to ATP hydrolysis by the reverse operation of the mitochondrial F_oF₁-ATPase, (ii) molecular and thermodynamic factors influencing the directionality of the F_oF₁-ATPase, (iii) the role of the adenine nucleotide translocase as the intermediary adenine nucleotide flux pathway between the cytosol and the mitochondrial matrix when mitochondria become ATP consumers, (iv) the role of the permeability transition pore in bypassing the ANT, thereby allowing the flux of ATP directly to the hydrolyzing F_oF₁-ATPase, (v) the impact of the permeability transition pore on glycolytic ATP production, and (vi) endogenous and exogenous interventions for limiting ATP hydrolysis by the mitochondrial F_oF₁-ATPase.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G₁, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent K_m for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower K_m constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.