The ABC of auxin transport: the role of p-glycoproteins in plant development

A surprising outcome of the Arabidopsis genome project was the annotation of a large number of sequences encoding members of the ABC transporter superfamily, including 22 genes encoding the p-glycoprotein (PGP) subfamily. As mammalian PGP orthologs are associated with multiple drug resistance, plant PGPs were initially presumed to function in detoxification, but were soon seen to have a developmental role. Here, we summarise recent studies of plant PGPs indicating that PGPs mediate the cellular and long-distance transport of the plant hormone auxin. One class of PGPs, represented by AtPGP1, catalyze auxin export, while another class with at least one member, AtPGP4, appears to function in auxin import. Current models on the physiological role of PGPs, their functional interaction and their involvement in cell-to cell (polar) auxin transport are discussed.     

The role of multiple partners in a digestive mutualism with a protocarnivorous plant

Background and aims

The protocarnivorous plant Paepalanthus bromelioides (Eriocaulaceae) is similar to bromeliads in that this plant has a rosette-like structure that allows rainwater to accumulate in leaf axils (i.e. phytotelmata). Although the rosettes of P. bromelioides are commonly inhabited by predators (e.g. spiders), their roots are wrapped by a cylindrical termite mound that grows beneath the rosette. In this study it is predicted that these plants can derive nutrients from recycling processes carried out by termites and from predation events that take place inside the rosette. It is also predicted that bacteria living in phytotelmata can accelerate nutrient cycling derived from predators.

Methods

The predictions were tested by surveying plants and animals, and also by performing field experiments in rocky fields from Serra do Cipó, Brazil, using natural abundance and enriched isotopes of ¹⁵N. Laboratory bioassays were also conducted to test proteolytic activities of bacteria from P. bromelioides rosettes.

Key Results

Analyses of ¹⁵N in natural nitrogen abundances showed that the isotopic signature of P. bromelioides is similar to that of carnivorous plants and higher than that of non-carnivorous plants in the study area. Linear mixing models showed that predatory activities on the rosettes (i.e. spider faeces and prey carcass) resulted in overall nitrogen contributions of 26·5 % (a top-down flux). Although nitrogen flux was not detected from termites to plants via decomposition of labelled cardboard, the data on ¹⁵N in natural nitrogen abundance indicated that 67 % of nitrogen from P. bromelioides is derived from termites (a bottom-up flux). Bacteria did not affect nutrient cycling or nitrogen uptake from prey carcasses and spider faeces.

Conclusions

The results suggest that P. bromelioides derive nitrogen from associated predators and termites, despite differences in nitrogen cycling velocities, which seem to have been higher in nitrogen derived from predators (leaves) than from termites (roots). This is the first study that demonstrates partitioning effects from multiple partners in a digestion-based mutualism. Despite most of the nitrogen being absorbed through their roots (via termites), P. bromelioides has all the attributes necessary to be considered as a carnivorous plant in the context of digestive mutualism.     

Calcium ions are involved in the delay of plant cell cycle progression by abiotic stresses

Higher plants respond to environmental stresses by a sequence of reactions which include the reduction of growth by affecting cell division. It has been shown that calcium ions plays a role as a second messenger in mediating various defence responses under environmental stresses. In this study, the role of calcium ions on cell cycle progression under abiotic stresses has been examined in tobacco BY-2 suspension culture cells. Using synchronized BY-2 cells expressing the endogenous calcium sensor aequorin as experimental system, we could show that oxidative and hypoosmotic stress both induce an increase of intracellular calcium and cause a delay of the cell cycle. The inhibitory effect of these abiotic stress stimuli on cell cycle progression could be mimicked by increasing the intracellular calcium concentration via application of an external electrical field. Likewise, depletion of calcium ions in the culture medium suppressed the effect of the stimuli tested. These results demonstrate that calcium signalling is involved in the regulation of cell cycle progression in response to abiotic stress.     

The effect of testosterone on ascorbic acid synthesis in rooster comb

The level of “total” ascorbic acid (ascorbate+dehydroascorbate) has been measured in the mucoid layer of combs from normal roosters, capons and capons treated with testosterone. The “total” ascorbate level in capon comb was lower than the value obtained from combs from normal roosters. This value returned towards normal in combs from capons treated with testosterone. The specific activity of L-gulonate-NADP⁺ oxidoreductase, an enzyme in the pathway of ascorbate biosynthesis, also was measured. The specific activity levels followed a pattern similar to the ascorbate levels in the three types of combs utilized. The results are consistent with the possible role of L-ascorbic acid as a cofactor in the synthesis of collagen, a process which also appears to be dependent on the level of testosterone in the comb mucoid layer.     

The effect of calcium ions on subcellular localization of aldolase-FBPase complex in skeletal muscle

In skeletal muscles, FBPase-aldolase complex is located on alpha-actinin of the Z-line. In the present paper, we show evidence that stability of the complex is regulated by calcium ions. Real time interaction analysis, confocal microscopy and the protein exchange method have revealed that elevated calcium concentration decreases association constant of FBPase-aldolase and FBPase-alpha-actinin complex, causes fast dissociation of FBPase from the Z-line and slow accumulation of aldolase within the I-band and M-line. Therefore, the release of Ca2+ during muscle contraction might result, simultaneously, in the inhibition of glyconeogenesis and in the acceleration of glycolysis.     

Methyl jasmonate-induced lateral root formation in rice: The role of heme oxygenase and calcium

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca²⁺ chelators and Ca²⁺ channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca²⁺ may regulate MJ action mainly through CaM-dependent mechanism.     

The role of RGS protein in agonist-dependent relaxation of GIRK currents in Xenopus oocytes

G protein coupled inward rectifier K⁺ channels (GIRK) are activated by the G_βγ subunits of G proteins upon activation of G protein coupled receptors (GPCRs). Receptor-stimulated GIRK currents are known to possess a curious property, termed “agonist-dependent relaxation,” denoting a slow current increase upon stepping the membrane voltage from positive to negative potentials. Regulators of G protein signaling (RGS) proteins have earlier been implicated in this phenomenon since RGS coexpression was required for relaxation to be observed in heterologous expression systems. However, a recent study presented contrasting evidence that GIRK current relaxation reflects voltage sensitive agonist binding to the GPCR. The present study re-examined the role of RGS protein in agonist-dependent relaxation and found that RGS coexpression is not necessary for the relaxation phenomenon. However, RGS4 speeds up relaxation kinetics, allowing the phenomenon to be observed using shorter voltage steps. These findings resolve the controversy regarding the role of RGS protein vs. GPCR voltage sensitivity in mediating agonist-dependent relaxation of GIRK currents.     

The role of organ of Corti mass in passive cochlear tuning

The mechanism for passive cochlear tuning remains unsettled. Early models considered the organ of Corti complex (OCC) as a succession of spring-mass resonators. Later, traveling wave models showed that passive tuning could arise through the interaction of cochlear fluid mass and OCC stiffness without local resonators. However, including enough OCC mass to produce local resonance enhanced the tuning by slowing and thereby growing the traveling wave as it approached its resonant segment. To decide whether the OCC mass plays a role in tuning, the frequency variation of the wavenumber of the cochlear traveling wave was measured (in vivo, passive cochleae) and compared to theoretical predictions. The experimental wavenumber was found by taking the phase difference of basilar membrane motion between two longitudinally spaced locations and dividing by the distance between them. The theoretical wavenumber was a solution of the dispersion relation of a three-dimensional cochlear model with OCC mass and stiffness as the free parameters. The experimental data were only well fit by a model that included OCC mass. However, as the measurement position moved from a best-frequency place of 40 to 12 kHz, the role of mass was diminished. The notion of local resonance seems to only apply in the very high-frequency region of the cochlea.     

The role of N-glycosylation in transport function and surface targeting of the human solute carrier PAT1

In the present study we show in the Xenopus laevis expression system that the proton-coupled amino acid transporter 1 (PAT1, SLC36A1) is glycosylated at asparagine residues N174, N183 and N470. To determine the functional role of N-glycosylation, glycosylation-deficient mutants were analyzed by two-electrode voltage-clamp measurements after expression in X. laevis oocytes. Single replacements of asparagine residues had no effect on transport activity. However, multiple substitutions resulted in a decreased transport rate, leaving K_t unchanged. Immunofluorescence localisation revealed a diminished plasma membrane expression of glycosylation-defective mutants. This indicates that N-glycans are not required for transport function, but are important for membrane targeting.     

Putative role of the adenosine A3 receptor in the antiproliferative action of N 6-(2-isopentenyl)adenosine

We tested a panel of naturally occurring nucleosides for their affinity towards adenosine receptors. Both N ⁶-(2-isopentenyl)adenosine (IPA) and racemic zeatin riboside were shown to be selective human adenosine A₃ receptor (hA₃R) ligands with affinities in the high nanomolar range (K _i values of 159 and 649 nM, respectively). These values were comparable to the observed K _i value of adenosine on hA₃R, which was 847 nM in the same radioligand binding assay. IPA also bound with micromolar affinity to the rat A₃R. In a functional assay in Chinese hamster ovary cells transfected with hA₃R, IPA and zeatin riboside inhibited forskolin-induced cAMP formation at micromolar potencies. The effect of IPA could be blocked by the A₃R antagonist VUF5574. Both IPA and reference A₃R agonist 2-chloro-N ⁶-(3-iodobenzyl)adenosine-5′-N-methylcarboxamide (Cl-IB-MECA) have known antitumor effects. We demonstrated strong and highly similar antiproliferative effects of IPA and Cl-IB-MECA on human and rat tumor cell lines LNCaP and N1S1. Importantly, the antiproliferative effect of low concentrations of IPA on LNCaP cells could be fully blocked by the selective A₃R antagonist MRS1523. At higher concentrations, IPA appeared to inhibit cell growth by an A₃R-independent mechanism, as was previously reported for other A₃R agonists. We used HPLC to investigate the presence of endogenous IPA in rat muscle tissue, but we could not detect the compound. In conclusion, the antiproliferative effects of the naturally occurring nucleoside IPA are at least in part mediated by the A₃R.     

Characterisation of the interface between nucleophosmin (NPM) and p53: potential role in p53 stabilisation

We have used surface plasmon resonance to quantify the kinetics and stoichiometry of the interaction between p53 and nucleophosmin (NPM). Domains characterising the interface between the two proteins were identified by chemical cross-linking, proteolytic digestion and mass spectrometry based peptide mapping.We show that the C-terminal domain of NPM (residues 242-269) interacts with two regions of p53 (residues 175-196 and residues 343-363) which belong, respectively, to the DNA binding domain and the tetramerisation domain. Potential biological consequences of such interactions are discussed.     

Expression and functional role of formyl peptide receptor in human bone marrow-derived mesenchymal stem cells

We investigated the expression of formyl peptide receptor (FPR) and its functional role in human bone marrow-derived mesenchymal stem cells (MSCs). We analyzed the expression of FPR by using ligand-binding assay with radio-labeled N-formyl-met-leu-phe (fMLF), and found that MSCs express FPR. FMLF stimulated intracellular calcium increase, mitogen-activated protein kinases activation, and Akt activation, which were mediated by G(i) proteins. MSCs were chemotactically migrated to fMLF. FMLF-induced MSC chemotaxis was also completely inhibited by pertussis toxin, LY294002, and PD98059, indicating the role of G(i) proteins, phosphoinositide 3-kinase, and extracellular signal regulated protein kinase. N-terminal fragment of annexin-1, Anx-1(2-26), an endogenous agonist for FPR, also induced chemotactic migration of MSCs. Thus MSCs express functional FPR, suggesting a new (patho)physiological role of FPR and its ligands in regulating MSC trafficking during induction of injured tissue repair.     

Structural and Kinetic Effects of PAK3 Phosphorylation Mimic of cTnI(S151E) on the cTnC-cTnI Interaction in the Cardiac Thin Filament

Residue Ser151 of cardiac troponin I (cTnI) is known to be phosphorylated by p21-activated kinase 3 (PAK3). It has been found that PAK3-mediated phosphorylation of cTnI induces an increase in the sensitivity of myofilament to Ca²⁺, but the detailed mechanism is unknown. We investigated how the structural and kinetic effects mediated by pseudo-phosphorylation of cTnI (S151E) modulates Ca²⁺-induced activation of cardiac thin filaments. Using steady-state, time-resolved Förster resonance energy transfer (FRET) and stopped-flow kinetic measurements, we monitored Ca²⁺-induced changes in cTnI-cTnC interactions. Measurements were done using reconstituted thin filaments, which contained the pseudo-phosphorylated cTnI(S151E). We hypothesized that the thin filament regulation is modulated by altered cTnC-cTnI interactions due to charge modification caused by the phosphorylation of Ser151 in cTnI. Our results showed that the pseudo-phosphorylation of cTnI (S151E) sensitizes structural changes to Ca²⁺ by shortening the intersite distances between cTnC and cTnI. Furthermore, kinetic rates of Ca²⁺ dissociation-induced structural change in the regulatory region of cTnI were reduced significantly by cTnI (S151E). The aforementioned effects of pseudo-phosphorylation of cTnI were similar to those of strong crossbridges on structural changes in cTnI. Our results provide novel information on how cardiac thin filament regulation is modulated by PAK3 phosphorylation of cTnI.     

Mammalian cell genetics. 3. Characterization of x-ray-induced forward mutations in Chinese hamster cell cultures

X-irradiation induces forward mutations from 8-azaguanine sensitvity to resistance in Chinese hamster cells in culture. At this locus the number of induced mutations increases non-linearly with X-ray exposure. The mutation rate increase from 4.2·10⁻⁷ per locus per R with 200 R to 1.8·10⁻⁶ per locus per R with 1200 R. Several factors including cell density markedly influence the mutational yield. Reversion tests using specific chemical mutagens on 72 randomly isolated, azaguanine-resistant mutants suggest that both point mutations and chromosome deletions might have occurred in the hamster cells after exposure to ionizing radiation.     

Antioxidant and phenolic acid profiles of tissue cultured and acclimatized Merwilla plumbea plantlets in relation to the applied cytokinins

Merwilla plumbea (Lindl.) Speta is an important medicinal plant widely used in traditional medicine. We evaluated the effect of five cytokinins [benzyladenine (BA), 2-isopentenyladenine (2iP), meta-topolin (mT), meta-topolin riboside (mTR), and meta-methoxy-9-tetrahydropyran-2-yl-topolin (MemTTHP)] on the level of phenolic acids and antioxidant activity of M. plumbea during the tissue culture and acclimatization stages. Two cytokinins (mT and mTR) significantly improved the antioxidant activity of tissue culture plantlets while the control plantlets were better after acclimatization. Using UPLC–MS/MS, the levels of hydroxybenzoic and hydroxycinnamic acid derivatives (phenolic acids) varied significantly during tissue culture and acclimatization, depending on the cytokinin and plant part analyzed. Vanillic acid (24.9 μg g⁻¹) detected in underground parts of tissue culture plants supplemented with BA was the most abundant phenolic acid detected. The current findings indicate that the phytochemicals together with the bioactivity during in vitro propagation of M. plumbea is influenced by the cytokinin type used and the stage of plant material collection.     

The role of electrostatic interactions in calmodulin-peptide complex formation

The complex between calmodulin and the calmodulin-binding portion of smMLCKp has been studied. Electrostatic interactions have been anticipated to be important in this system where a strongly negative protein binds a peptide with high positive charge. Electrostatic interactions were probed by varying the pH in the range from 4 to 11 and by charge deletions in CaM and smMLCKp. The change in net charge of CaM from approximately -5 at pH 4.5 to -15 at pH 7.5 leaves the binding constant virtually unchanged. The affinity was also unaffected by mutations in CaM and charge substitutions in the peptide. The insensitivity of the binding constant to pH may seem surprising, but it is a consequence of the high charge on both protein and peptide. At low pH it is further attenuated by a charge regulation mechanism. That is, the protein releases a number of protons when binding the positively charged peptide. We speculate that the role of electrostatic interactions is to discriminate against unbound proteins rather than to increase the affinity for any particular target protein.     

The role of nitric oxide in the germination of plant seeds and pollen

Two complex physiological processes, with opposite positions in the plant's life-cycle, seed and pollen germination, are vital to the accomplishment of successful plant growth and reproduction. This review summarizes the current state of knowledge of the intersection of NO signalling with the signalling pathways of ABA, GA, and ethylene; plant hormones that control the release of plant seeds from dormancy and germination. The cross-talk of NO and ROS is involved in the light- and hormone-specific regulation of seeds’ developmental processes during the initiation of plant ontogenesis. Similarly to seed germination, the mechanisms of plant pollen hydration, germination, tube growth, as well as pollen-stigma recognition are tightly linked to the proper adjustment of NO and ROS levels. The interaction of NO with ROS and secondary messengers such as Ca²⁺, cAMP and cGMP discovered in pollen represent a common mechanism of NO signalling. The involvement of NO in both breakpoints of plant physiology, as well as in the germination of spores within fungi and oomycetes, points toward NO as a component of an evolutionary conserved signalling pathway.     

Cross-linking of transmembrane helices in proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli: implications for the structure and function of the membrane domain

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α and a β subunit of 54 and 49 kDa, respectively, and is made up of three domains. Domain I (dI) and III (dIII) are hydrophilic and contain the NAD(H)- and NADP(H)-binding sites, respectively, whereas the hydrophobic domain II (dII) contains 13 transmembrane α-helices and harbours the proton channel. Using a cysteine-free transhydrogenase, the organization of dII and helix-helix distances were investigated by the introduction of one or two cysteines in helix-helix loops on the periplasmic side. Mutants were subsequently cross-linked in the absence and presence of diamide and the bifunctional maleimide cross-linker o-PDM (6 Å), and visualized by SDS-PAGE.In the α₂β₂ tetramer, αβ cross-links were obtained with the αG476C-βS2C, αG476C-βT54C and αG476C-βS183C double mutants. Significant αα cross-links were obtained with the αG476C single mutant in the loop connecting helix 3 and 4, whereas ββ cross-links were obtained with the βS2C, βT54C and βS183C single mutants in the beginning of helix 6, the loop between helix 7 and 8 and the loop connecting helix 11 and 12, respectively. In a model based on 13 mutants, the interface between the α and β subunits in the dimer is lined along an axis formed by helices 3 and 4 from the α subunit and helices 6, 7 and 8 from the β subunit. In addition, helices 2 and 4 in the α subunit together with helices 6 and 12 in the β subunit interact with their counterparts in the α₂β₂ tetramer. Each β subunit in the α₂β₂ tetramer was concluded to contain a proton channel composed of the highly conserved helices 9, 10, 13 and 14.     

The role of frataxin in fission yeast iron metabolism: Implications for Friedreich's ataxia

Background

The neurodegenerative disease Friedreich's ataxia is the result of frataxin deficiency. Frataxin is a mitochondrial protein involved in iron–sulfur cluster (Fe–S) cofactor biogenesis, but its functional role in this pathway is debated. This is due to the interconnectivity of iron metabolic and oxidative stress response pathways that make distinguishing primary effects of frataxin deficiency challenging. Since Fe–S cluster assembly is conserved, frataxin overexpression phenotypes in a simple eukaryotic organism will provide additional insight into frataxin function.

Methods

The Schizosaccharomyces pombe frataxin homologue (fxn1) was overexpressed from a plasmid under a thiamine repressible promoter. The S. pombe transformants were characterized at several expression strengths for cellular growth, mitochondrial organization, iron levels, oxidative stress, and activities of Fe–S cluster containing enzymes.

Results

Observed phenotypes were dependent on the amount of Fxn1 overexpression. High Fxn1 overexpression severely inhibited S. pombe growth, impaired mitochondrial membrane integrity and cellular respiration, and led to Fxn1 aggregation. Cellular iron accumulation was observed at moderate Fxn1 overexpression but was most pronounced at high levels of Fxn1. All levels of Fxn1 overexpression up-regulated oxidative stress defense and mitochondrial Fe–S cluster containing enzyme activities.

Conclusions

Despite the presence of oxidative stress and accumulated iron, activation of Fe–S cluster enzymes was common to all levels of Fxn1 overexpression; therefore, Fxn1 may regulate the efficiency of Fe–S cluster biogenesis in S. pombe.

General Significance

We provide evidence that suggests that dysregulated Fe–S cluster biogenesis is a primary effect of both frataxin overexpression and deficiency as in Friedreich's ataxia.     

Localized activation of Src-family protein kinases in the mouse egg

Recent studies in species that fertilize externally have demonstrated that fertilization triggers localized activation of Src-family protein kinases in the egg cortex. However, the requirement for Src-family kinases in activation of the mammalian egg is different from lower species and the objective of this study was to characterize changes in the distribution and activity of Src-family protein tyrosine kinases (PTKs) during zygotic development in the mouse. Immunofluorescence analysis of mouse oocytes and zygotes with an anti-phosphotyrosine antibody revealed that fertilization stimulated accumulation of P-Tyr-containing proteins in the egg cortex and that their abundance was elevated in the region overlying the MII spindle. In addition, the poles of the MII spindle exhibited elevated P-Tyr levels. As polar body extrusion progressed, P-Tyr-containing proteins were especially concentrated in the region of cortex adjacent to the maternal chromatin and the forming polar body. In contrast, P-Tyr labeling of the spindle poles eventually disappeared as meiosis II progressed to anaphase II. In approximately 24% of cases, the fertilizing sperm nucleus was associated with increased P-Tyr labeling in the overlying cortex and oolemma. To determine whether Src-family protein tyrosine kinases could be responsible for the observed changes in the distribution of P-Tyr containing proteins, an antibody to the activated form of Src-family PTKs was used to localize activated Src, Fyn or Yes. Activated Src-family kinases were found to be strongly associated with the meiotic spindle at all stages of meiosis II; however, no concentration of labeling was evident at the egg cortex. The absence of cortical Src-family PTK activity continued until the blastocyst stage when strong cortical activity became evident. At the pronuclear stage, activated Src-family PTKs became concentrated around the pronuclei in close association with the nuclear envelope. This pattern was unique to the earliest stages of development and disappeared by the eight cell stage. Functional studies using chemical inhibitors and a dominant-negative Fyn construct demonstrated that Src-family PTKs play an essential role in completion of meiosis II following fertilization and progression from the pronuclear stage into mitosis. These data suggest that while Src-family PTKs are not required for fertilization-induced calcium oscillations, they do play a critical role in development of the zygote. Furthermore, activation of these kinases in the mouse egg is limited to distinct regions and occurs at specific times after fertilization.