Experimental investigation of the origin of fynbos plant community structure after fire

Background and aims

Species in plant communities segregate along fine-scale hydrological gradients. Although this phenomenon is not unique to fynbos, this community regenerates after fire and therefore provides an opportunity to study the ecological genesis of hydrological niche segregation.

Methods

Following wildfires at two field sites where we had previously mapped the vegetation and monitored the hydrology, seeds were moved experimentally in >2500 intact soil cores up and down soil-moisture gradients to test the hypothesis that hydrological niche segregation is established during the seedling phase of the life cycle. Seedling numbers and growth were then monitored and they were identified using DNA bar-coding, the first use of this technology for an experiment of this kind.

Key Results

At the site where niche segregation among Restionaceae had previously been found, the size of seedlings was significantly greater, the wetter the location into which they were moved, regardless of the soil moisture status of their location of origin, or of the species. Seedling weight was also significantly greater in a competition treatment where the roots of other species were excluded. No such effects were detected at the control site where niche segregation among Restionaceae was previously found to be absent.

Conclusions

The finding that seedling growth on hydrological gradients in the field is affected by soil moisture status and by root competition shows that hydrological niche segregation could potentially originate in the seedling stage. The methodology, applied at a larger scale and followed-through for a longer period, could be used to determine whether species are differently affected by soil moisture.     

The Tat System for Membrane Translocation of Folded Proteins Recruits the Membrane-stabilizing Psp Machinery in Escherichia coli

Tat systems transport folded proteins across energized membranes of bacteria, archaea, and plant plastids. In Escherichia coli, TatBC complexes recognize the transported proteins, and TatA complexes are recruited to facilitate transport. We achieved an abstraction of TatA from membranes without use of detergents and observed a co-purification of PspA, a membrane-stress response protein. The N-terminal transmembrane domain of TatA was required for the interaction. Electron microscopy displayed TatA complexes in direct contact with PspA. PspB and PspC were important for the TatA-PspA contact. The activator protein PspF was not involved in the PspA-TatA interaction, demonstrating that basal levels of PspA already interact with TatA. Elevated TatA levels caused membrane stress that induced a strictly PspBC- and PspF-dependent up-regulation of PspA. TatA complexes were found to destabilize membranes under these conditions. At native TatA levels, PspA deficiency clearly affected anaerobic TMAO respiratory growth, suggesting that energetic costs for transport of large Tat substrates such as TMAO reductase can become growth limiting in the absence of PspA. The physiological role of PspA recruitment to TatA may therefore be the control of membrane stress at active translocons.     

Specific residues of a conserved domain in the N terminus of the human cytomegalovirus pUL50 protein determine its intranuclear interaction with pUL53

Herpesviral capsids are assembled in the host cell nucleus and are subsequently translocated to the cytoplasm. During this process it has been demonstrated that the human cytomegalovirus proteins pUL50 and pUL53 interact and form, together with other viral and cellular proteins, the nuclear egress complex at the nuclear envelope. In this study we provide evidence that specific residues of a conserved N-terminal region of pUL50 determine its intranuclear interaction with pUL53. In silico evaluation and biophysical analyses suggested that the conserved region forms a regular secondary structure adopting a globular fold. Importantly, site-directed replacement of individual amino acids by alanine indicated a strong functional influence of specific residues inside this globular domain. In particular, mutation of the widely conserved residues Glu-56 or Tyr-57 led to a loss of interaction with pUL53. Consistent with the loss of binding properties, mutants E56A and Y57A showed a defective function in the recruitment of pUL53 to the nuclear envelope in expression plasmid-transfected and human cytomegalovirus-infected cells. In addition, in silico analysis suggested that residues 3-20 form an amphipathic α-helix that appears to be conserved among Herpesviridae. Point mutants revealed a structural role of this N-terminal α-helix for pUL50 stability rather than a direct role in the binding of pUL53. In contrast, the central part of the globular domain including Glu-56 and Tyr-57 is directly responsible for the functional interaction with pUL53 and thus determines formation of the basic nuclear egress complex.     

A novel nuclear trafficking module regulates the nucleocytoplasmic localization of the rabies virus interferon antagonist, p protein

Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1-P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3-P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection.     

Dynamic disulfide scanning of the membrane-inserting Pf3 coat protein reveals multiple YidC substrate contacts

The membrane insertase YidC inserts newly synthesized proteins into the plasma membrane. While defects in YidC homologs in animals and plants cause diseases, YidC in bacteria is essential for life. Membrane insertion and assembly of ATP synthase and respiratory complexes is catalyzed by YidC. To investigate how YidC interacts with membrane-inserting proteins, we generated single cysteine mutants in YidC and in the model substrate Pf3 coat protein. The single cysteine mutants were expressed and analyzed for disulfide formation during 30 s of synthesis. The results show that the substrate contacts different YidC residues in four of the six transmembrane regions. The residues are located either in the region of the inner leaflet, in the center, as well as in the periplasmic leaflet, consistent with the hypothesis that YidC presents a hydrophobic platform for inserting membrane proteins. In a YidC mutant where most of the contacting residues were mutated to serines, YidC function was severely disturbed and no longer active in a complementation test, suggesting that the residues are important for function. In addition, a Pf3 mutant with a defect in membrane insertion was deficient to contact the periplasmic residues of YidC.     

Direct plant gene delivery with a poly(amidoamine) dendrimer

Plant gene delivery is challenging due to the presence of plant cell walls. Conventional means such as Agrobacterium infection, biolistic particle bombardment, electroporation, or polyethylene glycol attachment are often characterized by high cost, labor extensiveness, and a significant perturbation to the growth of cells. We have succeeded in delivering GFP-encoding plasmid DNA to turfgrass cells using poly(amidoamine) dendrimers. Our new scheme utilizes the physiochemical properties as well as the nanosize of the poly(amidoamine) dendrimer for direct and noninvasive gene delivery. The GFP gene was expressed in the plant cells as observed by confocal fluorescence microscopy. The transfection efficiency may be further improved by optimizing the pH of the cell culture medium and the molar ratio of the dendrimer to DNA. The use of the current delivery system can be extended to virtually all plant species having successful regeneration systems in place.     

Effect of oral diazepam on feeding behavior and activity of Hawai’i ’amakihi (Hemignathus virens)

Feeding behavior and activity during captivity were studied in wild-caught Hawai’i ’amakihi, Hemignathus virens, to evaluate diazepam's hyperphagic and anxiolytic effects. Birds were captured in mist nets, given either oral diazepam (1 mg/kg) or an equivalent volume per weight of lactated Ringer's solution orally, and held in captivity for 6 h. Thirteen-minute focal animal samples were videotaped at the beginning of each hour. Feeding behaviors, grooming and picking events, changes in position, and body weights were recorded. Mean duration of feeding, percentage of time spent feeding, and number of feeding events were significantly higher for treatment birds than for controls, and significantly increased over time. Feeding duration was significantly correlated to weight change. Weight change was not significantly different between groups, but on average treatment birds lost less weight than control birds. No significant differences in grooming behaviors were found between the groups, but there was a session effect of increased grooming over time in both groups. Also, a significant session effect in movement events was apparent, with control birds becoming less active and treatment birds becoming more active over time. Results indicate diazepam increased feeding behaviors and movement in this passerine species during a short period of captivity.     

Test of soil extractants for their suitability in predicting Ca/Sr ratios in leaves and stems of sugar maple seedlings

Differential uptake and translocation of Ca and Sr in organisms have been reported, calling into question the use of Sr to track Ca cycling in the environment. We investigated the relationship between Ca/Sr ratios in soil extracts of various strengths (H₂O, NH₄Cl, and NH₄EDTA) and seedlings of sugar maple (Acer saccharum Marsh.) grown from natural regeneration on 37 sites. Our objectives were to determine if Ca/Sr ratios in soil extracts are correlated with those in sugar maple tissues, and what soil extractant best duplicate plant tissue Ca/Sr ratios. Leaves had higher Ca/Sr ratios than stems and the extractants did not produce equal Ca/Sr ratios: H₂O had the lowest Ca/Sr, and NH₄EDTA the highest. The relationships between soil extract Ca/Sr ratios and leaf and stem Ca/Sr ratios were significant and linear, but the slopes differed among extractants. The lowest slope (0.45) was observed for the water extract/leaves and the highest (2.15) for the NH₄EDTA extract/stem with discrimination factors ranging from 0.22 with NH₄EDTA to 1.59 for water. Leaf extracts were more strongly correlated with soil Ca/Sr than stem extracts (R ² of 0.57–0.7 vs. R ² of 0.45–0.6, respectively). These findings support the use of Ca/Sr ratios in plants to track their source of soil Ca, but they highlight the need to calibrate the relationships for the plant tissue and soil extractant used.