Evidence for nucleotide-dependent processes in the thylakoid lumen of plant chloroplasts - an update

The thylakoid lumen is an aqueous chloroplast compartment enclosed by the thylakoid membrane network. Bioinformatic and proteomic studies indicated the existence of 80-90 thylakoid lumenal proteins in Arabidopsis thaliana, having photosynthetic, non-photosynthetic or unclassified functions. None of the identified lumenal proteins had canonical nucleotide-binding motifs. It was therefore suggested that, in contrast to the chloroplast stroma harboring nucleotide-dependent enzymes and other proteins, the thylakoid lumen is a nucleotide-free compartment. Based on recent findings, we provide here an updated view about the presence of nucleotides in the thylakoid lumen of plant chloroplasts, and their role in function and dynamics of photosynthetic complexes.     

Subunit F modulates ATP binding and migration in the nucleotide-binding subunit B of the A1AO ATP synthase of Methanosarcina mazei Gö1

The interaction of the nucleotide-binding subunit B with subunit F is essential in coupling of ion pumping and ATP synthesis in A₁A_O ATP synthases. Here we provide structural and thermodynamic insights on the nucleotide binding to the surface of subunits B and F of Methanosarcina mazei Gö1 A₁A_O ATP synthase, which initiated migration to its final binding pocket via two transitional intermediates on the surface of subunit B. NMR- and fluorescence spectroscopy as well as ITC data combined with molecular dynamics simulations of the nucleotide bound subunit B and nucleotide bound B-F complex in explicit solvent, suggests that subunit F is critical for the migration to and eventual occupancy of the final binding site by the nucleotide of subunit B. Rotation of the C-terminus and conformational changes in subunit B are initiated upon binding with subunit F causing a perturbation that leads to the migration of ATP from the transition site 1 through an intermediate transition site 2 to the final binding site 3. This mechanism is elucidated on the basis of change in binding affinity for the nucleotide at the specific sites on subunit B upon complexation with subunit F. The change in enthalpy is further explained based on the fluctuating local environment around the binding sites.     

Design and dosimetric analysis of a 385 MHz TETRA head exposure system for use in human provocation studies

A new head exposure system for double‐blind provocation studies investigating possible effects of terrestrial trunked radio (TETRA)‐like exposure (385 MHz) on central nervous processes was developed and dosimetrically analyzed. The exposure system allows localized exposure in the temporal brain, similar to the case of operating a TETRA handset at the ear. The system and antenna concept enables exposure during wake and sleep states while an electroencephalogram (EEG) is recorded. The dosimetric assessment and uncertainty analysis yield high efficiency of 14 W/kg per Watt of accepted antenna input power due to an optimized antenna directly worn on the subject's head. Beside sham exposure, high and low exposure at 6 and 1.5 W/kg (in terms of maxSAR10g in the head) were implemented. Double‐blind control and monitoring of exposure is enabled by easy‐to‐use control software. Exposure uncertainty was rigorously evaluated using finite‐difference time‐domain (FDTD)‐based computations, taking into account anatomical differences of the head, the physiological range of the dielectric tissue properties including effects of sweating on the antenna, possible influences of the EEG electrodes and cables, variations in antenna input reflection coefficients, and effects on the specific absorption rate (SAR) distribution due to unavoidable small variations in the antenna position. This analysis yielded a reasonable uncertainty of <±45% (max to min ratio of 4.2 dB) in terms of maxSAR10g in the head and a variability of <±60% (max to min ratio of 6 dB) in terms of mass‐averaged SAR in different brain regions, as demonstrated by a brain region‐specific absorption analysis. Bioelectromagnetics 33:594–603, 2012. © 2012 Wiley Periodicals, Inc.     

Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.     

The Stem Region of Premembrane Protein Plays an Important Role in the Virus Surface Protein Rearrangement during Dengue Maturation

Newly assembled dengue viruses (DENV) undergo maturation to become infectious particles. The maturation process involves major rearrangement of virus surface premembrane (prM) and envelope (E) proteins. The prM-E complexes on immature viruses are first assembled as trimeric spikes in the neutral pH environment of the endoplasmic reticulum. When the virus is transported to the low pH environment of the exosomes, these spikes rearrange into dimeric structures, which lie parallel to the virus lipid envelope. The proteins involved in driving this process are unknown. Previous cryoelectron microscopy studies of the mature DENV showed that the prM-stem region (residues 111–131) is membrane-associated and may interact with the E proteins. Here we investigated the prM-stem region in modulating the virus maturation process. The binding of the prM-stem region to the E protein was shown to increase significantly at low pH compared with neutral pH in ELISAs and surface plasmon resonance studies. In addition, the affinity of the prM-stem region for the liposome, as measured by fluorescence correlation spectroscopy, was also increased when pH is lowered. These results suggest that the prM-stem region forms a tight association with the virus membrane and attracts the associated E protein in the low pH environment of exosomes. This will lead to the surface protein rearrangement observed during maturation.     

The trafficking protein SYP121 of Arabidopsis connects programmed stomatal closure and K⁺ channel activity with vegetative growth

The vesicle‐trafficking protein SYP121 (SYR1/PEN1) was originally identified in association with ion channel control at the plasma membrane of stomatal guard cells, although stomata of the Arabidopsis syp121 loss‐of‐function mutant close normally in ABA and high Ca²⁺. We have now uncovered a set of stomatal phenotypes in the syp121 mutant that reduce CO₂ assimilation, slow vegetative growth and increase water use efficiency in the whole plant, conditional upon high light intensities and low relative humidity. Stomatal opening and the rise in stomatal transpiration of the mutant was delayed in the light and following Ca²⁺‐evoked closure, consistent with a constitutive form of so‐called programmed stomatal closure. Delayed reopening was observed in the syp121, but not in the syp122 mutant lacking the homologous gene product; the delay was rescued by complementation with wild‐type SYP121 and was phenocopied in wild‐type plants in the presence of the vesicle‐trafficking inhibitor Brefeldin A. K⁺ channel current that normally mediates K⁺ uptake for stomatal opening was suppressed in the syp121 mutant and, following closure, its recovery was slowed compared to guard cells of wild‐type plants. Evoked stomatal closure was accompanied by internalisation of GFP‐tagged KAT1 K⁺ channels in both wild‐type and syp121 mutant guard cells, but their subsequently recycling was slowed in the mutant. Our findings indicate that SYP121 facilitates stomatal reopening and they suggest that K⁺ channel traffic and recycling to the plasma membrane underpins the stress memory phenomenon of programmed closure in stomata. Additionally, they underline the significance of vesicle traffic for whole‐plant water use and biomass production, tying SYP121 function to guard cell membrane transport and stomatal control.     

How does drought stress influence the decomposition of plant litter with contrasting quality in a grassland ecosystem?

Background and aims

Plant litter quality and water availability both control decomposition. The interaction of both parameters was never studied. We used a grassland site, where litter of contrasting quality, i.e. green litter (fresh leaves; high quality) and brown litter (dead leaves, which underwent senescence but which are still attached to the plant; low quality), is returned to soil. Green and brown litter were exposed in the field under regular weather and drought conditions. The objective of this study was to evaluate the effect of drought on the decomposition of both litter types.

Methods

We incubated green and brown litter of three different grassland species (Lolium perenne, Festuca arundinacea and Dactylis glomerata) alone or as litter mixture (1/3 of each of the three grassland species) in litterbags for 28?weeks. Drought conditions were simulated by rainfall exclusion. After incubation, litter residues were analysed for C and nitrogen (N) content and stable isotope composition. Additionally, we determined the response of the lignin and carbohydrate signatures to the contrasting conditions.

Results

C decomposition kinetics of green and brown litter under drought conditions could be explained by two pools of contrasting turnover times. Drought decreased leaf litter C and N decomposition by more than 50% compared to regular weather conditions, mainly by strongly decreasing the decomposition rate constants. The lowest C decomposition occurred for mixtures of litter from all three grassland species. Brown litter showed on average 15% higher reduction in carbon decomposition than green litter following drought. Lignin content remained similar for green and brown litter after drought and regular weather conditions, while sugar content remained similar in green litter and decreased by 18% for brown litter under drought conditions.

Conclusions

Our results showed different response of decomposition of litter with contrasting quality to drought. Low quality brown litter is likely to be more affected than high quality green litter. Thus, litter quality must be taken into account, when assessing the effect of drought on decomposition.     

Systems Dynamic Modeling of a Guard Cell Cl? Channel Mutant Uncovers an Emergent Homeostatic Network Regulating Stomatal Transpiration

Stomata account for much of the 70% of global water usage associated with agriculture and have a profound impact on the water and carbon cycles of the world. Stomata have long been modeled mathematically, but until now, no systems analysis of a plant cell has yielded detail sufficient to guide phenotypic and mutational analysis. Here, we demonstrate the predictive power of a systems dynamic model in Arabidopsis (Arabidopsis thaliana) to explain the paradoxical suppression of channels that facilitate K⁺ uptake, slowing stomatal opening, by mutation of the SLAC1 anion channel, which mediates solute loss for closure. The model showed how anion accumulation in the mutant suppressed the H⁺ load on the cytosol and promoted Ca²⁺ influx to elevate cytosolic pH (pH_i) and free cytosolic Ca²⁺ concentration ([Ca2+]_i), in turn regulating the K⁺ channels. We have confirmed these predictions, measuring pH_i and [Ca2+]_i in vivo, and report that experimental manipulation of pH_i and [Ca2+]_i is sufficient to recover K⁺ channel activities and accelerate stomatal opening in the slac1 mutant. Thus, we uncover a previously unrecognized signaling network that ameliorates the effects of the slac1 mutant on transpiration by regulating the K⁺ channels. Additionally, these findings underscore the importance of H⁺-coupled anion transport for pH_i homeostasis.Guard cells surround stomatal pores in the epidermis of plant leaves and regulate pore aperture to balance the demands for CO₂ in photosynthesis with the need to conserve water by the plant. Transpiration through stomata accounts for much of the 70% of global water usage associated with agriculture, and it has a profound impact on the water and carbon cycles of the world (Gedney et al., 2006; Betts et al., 2007). Guard cells open the pore by transport and accumulation of osmotically active solutes, mainly K⁺ and Cl⁻ and the organic anion malate²⁻ (Mal), to drive water uptake and cell expansion. They close the pore by coordinating the release of these solutes through K⁺ and anion channels at the plasma membrane. The past half-century has generated a wealth of knowledge on guard cell transport, signaling, and homeostasis, resolving the properties of the major transport processes and metabolic pathways for osmotic solute uptake and accumulation, and many of the signaling pathways that control them (Blatt, 2000; Schroeder et al., 2001; McAinsh and Pittman, 2009; Hills et al., 2012). Even so, much of stomatal dynamics remains unresolved, especially how the entire network of transporters in guard cells works to modulate solute flux and how this network is integrated with organic acid metabolism (Wang and Blatt, 2011) to achieve a dynamic range of stomatal apertures.This gap in understanding is most evident in a number of often unexpected observations, many of which have led necessarily to ad hoc interpretations. Among these, recent studies highlighted a diurnal variation in the free cytosolic Ca²⁺ concentration ([Ca2+]_i), high in the daytime despite the activation of primary ion-exporting ATPases, and have been interpreted to require complex levels of regulation (Dodd et al., 2007). Other findings wholly defy intuitive explanation. For example, the tpk1 mutant of Arabidopsis (Arabidopsis thaliana) removes a major pathway for K⁺ flux across the tonoplast and suppresses stomatal closure, yet the mutant has no significant effect on cellular K⁺ content (Gobert et al., 2007). Similarly, the Arabidopsis clcc mutant eliminates the H⁺-Cl⁻ antiporter at the tonoplast; it affects Cl⁻ uptake, reduces vacuolar Cl⁻ content, and slows stomatal opening; however, counterintuitively, it also suppresses stomatal closure (Jossier et al., 2010). In work leading to this study, we observed that the slac1 anion channel mutant of Arabidopsis paradoxically profoundly alters the activities of the two predominant K⁺ channels at the guard cell plasma membrane. The SLAC1 anion channel is a major pathway for anion loss from the guard cells during stomatal closure (Negi et al., 2008; Vahisalu et al., 2008), and its mutation leads to incomplete and slowed closure of stomata in response to physiologically relevant signals of dark, high CO₂, and the water-stress hormone abscisic acid. Guard cells of the slac1 mutant accumulate substantially higher levels of Cl⁻, Mal, and also K⁺ when compared with guard cells of wild-type Arabidopsis (Negi et al., 2008). The latter observation is consistent with additional impacts on K⁺ transport; however, a straightforward explanation for these findings has not been not forthcoming.Quantitative systems analysis offers one approach to such problems. Efforts to model stomatal function generally have been driven by a “top-down” approach (Farquhar and Wong, 1984; Eamus and Shanahan, 2002) and have not incorporated detail essential to understanding the molecular and cellular mechanics that drive stomatal movement. Only recently we elaborated a quantitative systems dynamic approach to modeling the stomatal guard cell that incorporates all of the fundamental properties of the transporters at the plasma membrane and tonoplast, the salient features of osmolite metabolism, and the essential cytosolic pH (pH_i) and [Ca2+]_i buffering characteristics that have been described in the literature (Hills et al., 2012). The model resolved with this approach (Chen et al., 2012b) successfully recapitulated a wide range of known stomatal behaviors, including transport and aperture dependencies on extracellular pH, KCl, and CaCl₂ concentrations, diurnal changes in [Ca2+]_i (Dodd et al., 2007), and oscillations in membrane voltage and [Ca2+]_i thought to facilitate stomatal closure (Blatt, 2000; McAinsh and Pittman, 2009; Chen et al., 2012b). We have used this approach to resolve the mechanism behind the counterintuitive alterations in K⁺ channel activity uncovered in the slac1 mutant of Arabidopsis. Here, we show how anion accumulation in the mutant affects the H⁺ and Ca²⁺ loads on the cytosol, elevating pH_i and [Ca2+]_i, and in turn regulating the K⁺ channels. We have validated the key predictions of the model and, in so doing, have uncovered a previously unrecognized homeostatic network that ameliorates the effects of the slac1 mutant on transpiration from the plant.     

Path integration controls nest-plume following in desert ants

The desert ant Cataglyphis fortis is equipped with sophisticated navigational skills for returning to its nest after foraging. The ant's primary means for long-distance navigation is path integration, which provides a continuous readout of the ant's approximate distance and direction from the nest. The nest is pinpointed with the aid of visual and olfactory landmarks. Similar landmark cues help ants locate familiar food sites. Ants on their outward trip will position themselves so that they can move upwind using odor cues to find food. Here we show that homing ants also move upwind along nest-derived odor plumes to approach their nest. The ants only respond to odor plumes if the state of their path integrator tells them that they are near the nest. This influence of path integration is important because we could experimentally provoke ants to follow odor plumes from a foreign, conspecific nest and enter that nest. We identified CO(2) as one nest-plume component that can by itself induce plume following in homing ants. Taken together, the results suggest that path-integration information enables ants to avoid entering the wrong nest, where they would inevitably be killed by resident ants.