Rhagada revisited: on the taxonomy of species from the Kimberley and Dampierland,Western Australia (Pulmonata,Camaenidae)

The genus Rhagada is the second most diverse camaenid genus in Australia. We examined anatomical and mitochondrial characters of previously unidentified material from the Kimberley that was earmarked to potentially represent new species in recently published molecular phylogenetic studies. Our comparisons revealed that specimens from Gibbings Island (‘R. sp. Gibbings’) were morphologically and genetically most similar to Rhagada cygna from the Dampier Peninsula. Hence, ‘R. sp. Gibbings’ is considered to be identical to R. cygna. In addition, we found that R. cygna as so delimited is not clearly distinguished from the second species on the Dampier Peninsula, Rhagada bulgana. Both species differ rather subtly in anatomical and mitochondrial characters, indicating their close relationships and potentially incomplete evolutionary differentiation. Furthermore, we describe two new species based on comparative morphology and mitochondrial sequences: Rhagada worora n. sp. from the Prince Regent Reserve in the Kimberley and Rhagada karajarri n. sp. from Dampierland. The present study confirms that species in Rhagada are best identified by means of both morphological and molecular data.

http://zoobank.org/urn:lsid:zoobank.org:pub:556E1866-6F9E-4CC0-8ACF-CD56E929501F     

Tsunami impact on the population development of a critically endangered hummingbird species of a Pacific island

It is a long-standing question how tsunamis can influence wild populations of animals and plants. Here, we assessed short-term changes in the population of the critically endangered Juan Fernández Firecrown (Sephanoides fernandensis) by using abundance data recorded 1 year before and 1 year after the 2010 Chilean tsunami. We tested that the abundance of Firecrowns declined in the areas where the tsunami caused the massive loss of Cabbage Trees, an important seasonal nectar source for Firecrowns. The abundance of Juan Fernández Firecrowns decreased after the tsunami, but also was affected by the habitat type, altitude, and the abundance of Cabbage Trees. Firecrowns tended to be more abundant in settlement areas than in native forest whereas the reduction in Firecrown abundance after the tsunami was more intense in settlement areas than in native forest. As expected, this habitat effect was dependent on the massive loss of Cabbage Trees in settlement areas following the tsunami. In spite of the short-term nature of our data, our results are conclusive in showing that the loss of an important food source causes short-term changes in the distribution and abundance of Firecrowns, which, in turn, could contribute to population decline.     

Jasmonic acid perception by COI1 involves inositol polyphosphates in Arabidopsis thaliana

Plant responses to wounding are part of their defense responses against insects, and are tightly regulated. The isoleucin conjugate of jasmonic acid (JA‐Ile) is a major regulatory molecule. We have previously shown that inositol polyphosphate signals are required for defense responses in Arabidopsis; however, the way in which inositol polyphosphates contribute to plant responses to wounding has so far remained unclear. Arabidopsis F‐box proteins involved in the perception of JA‐Ile (COI1) and auxin (TIR1) are structurally similar. Because TIR1 has recently been shown to contain inositol hexakisphosphate (InsP₆) as a co‐factor of unknown function, here we explored the possibility that InsP₆ or another inositol polyphosphate is required for COI1 function. In support of this hypothesis, COI1 variants with changes in putative inositol polyphosphate coordinating residues exhibited a reduced interaction with the COI1 target, JAZ9, in yeast two‐hybrid tests. The equivalent COI1 variants displayed a reduced capability to rescue jasmonate‐mediated root growth inhibition or silique development in Arabidopsis coi1 mutants. Yeast two‐hybrid tests using wild‐type COI1 in an ipk1Δ yeast strain exhibiting increased levels of inositol pentakisphosphate (InsP₅) and reduced levels of InsP₆ indicate an enhanced COI1/JAZ9 interaction. Consistent with these findings, Arabidopsis ipk1‐1 mutants, also with increased InsP₅ and reduced InsP₆ levels, showed increased defensive capabilities via COI1‐mediated processes, including wound‐induced gene expression, defense against caterpillars or root growth inhibition by jasmonate. The combined data from experiments using mutated COI1 variants, as well as yeast and Arabidopsis backgrounds altered in inositol polyphosphate metabolism, indicate that an inositol polyphosphate, and probably InsP₅, contributes to COI1 function.     

Knockdown of CENH3 in Arabidopsis reduces mitotic divisions and causes sterility by disturbed meiotic chromosome segregation

The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP-CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP-CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP-CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Cellular aging leads to functional heterogeneity of hematopoietic stem cells: a modeling perspective

Hematopoietic stem cells (HSCs) are the source for the life-long supply of functional cells in peripheral blood while they simultaneously maintain their own reserve pool. However, there is accumulating evidence that HSCs are themselves subject to quantitative and qualitative exhaustion. Although several processes linked to mitotic activity can potentially account for the observed aging phenomena (e.g., DNA damage, telomere shortening, epigenetic modification), a precise understanding of HSC exhaustion is still missing. It is particularly unclear how individual aging processes on the single-cell level translate on the phenotypic level of the overall tissue and whether there is a functional implication of an age-structured HSC population. We address these issues by applying a novel mathematical model of HSC organization in which division-specific, cumulative alterations of stem cell quality determine the phenotypic and functional appearance of the overall cell population. Adapting the model to a number of basic experimental findings, we quantify the level of additional heterogeneity that is introduced by a population of individually aging cells. Based on this model, we are able to conclude that division-dependent processes of cellular aging explain a wide range of phenomena on HSC exhaustion and that HSC aging needs to be considered as a highly heterogeneous process. We furthermore report that functional heterogeneity between young and old HSCs appears closely similar to the phenomena described for long- and short-term repopulating cells. We speculate whether differential, division-coupled stem cell aging introduces an intra-animal variability that also accounts for heterogeneity with respect to the repopulation ability of HSCs.     

Detection of viral sequence fragments of HIV-1 subfamilies yet unknown

Background  

Methods of determining whether or not any particular HIV-1 sequence stems - completely or in part - from some unknown HIV-1 subtype are important for the design of vaccines and molecular detection systems, as well as for epidemiological monitoring. Nevertheless, a single algorithm only, the Branching Index (BI), has been developed for this task so far. Moving along the genome of a query sequence in a sliding window, the BI computes a ratio quantifying how closely the query sequence clusters with a subtype clade. In its current version, however, the BI does not provide predicted boundaries of unknown fragments.     

The K+ battery-regulating Arabidopsis K+ channel AKT2 is under the control of multiple post-translational steps

Potassium (K⁺) is an important nutrient for plants. It serves as a cofactor of various enzymes and as the major inorganic solute maintaining plant cell turgor. In a recent study, an as yet unknown role of K⁺ in plant homeostasis was shown. It was demonstrated that K⁺ gradients in vascular tissues can serve as an energy source for phloem (re)loading processes and that the voltage-gated K⁺ channels of the AKT2-type play a unique role in this process. The AKT2 channel can be converted by phosphorylation of specific serine residues (S210 and S329) into a non-rectifying channel that allows a rapid efflux of K⁺ from the sieve element/companion cells (SE/CC) complex. The energy of this flux is used by other transporters for phloem (re)loading processes. Nonetheless, the results do indicate that post-translational modifications at S210 and S329 alone cannot explain AKT2 regulation. Here, we discuss the existence of multiple post-translational modification steps that work in concert to convert AKT2 from an inward-rectifying into a non-rectifying K⁺ channel.Key words: potassium, channel, potassium channel, AKT2, phloem (re)loading, post-translational modifications, potassium batteryPotassium (K⁺) is the most abundant mineral element in plants, and together with nitrogen and phosphorous, is limiting for plant production in many natural and agricultural habitats. Voltage-gated K⁺ channels are key players in the acquisition of K⁺ ions from the soil and in its redistribution within the plant.¹ Structurally, these channels result from the assembly of four so-called α-subunits. The subunits are encoded by nine genes in Arabidopsis and both homo- and hetero-tetramers are expressed.^2,3 The K⁺ channel α-subunits can be categorized into four different subfamilies, based on the voltage-gating characteristics of the exogenous K⁺ conductance when expressed in an appropriate heterologous expression system. K_in α-subunits form hyperpolarization-activated channels that mediate K⁺ uptake.^4–7 K_out α-subunits form depolarization-activated channels that mediate K⁺ release from cells.^8–10 K_silent subunits appear unable to yield functional homomeric channels, but can combine with K_in subunits and fine-tune the K⁺-uptake properties of the resulting heteromeric channels.^11–14 Finally, K_weak α-subunits form channels with complex voltage-gating; they allow both K⁺ uptake and release.^15–19 In Arabidopsis, a single member is found in this subfamily, AKT2, and this channel can assemble in heteromeric channels with the K_in subunit KAT2.²⁰To date, only scarce and speculative information has been obtained for the function of K_weak channels. When expressed in heterologous expression systems, two different subpopulations of AKT2 channels differing in their sensitivity to voltage were found.²¹ Channels of the first type showed gating properties and currents analogous to that of K_in channels, while the other sort enabled a non-rectified (leak-like) current; they were open over the entire physiological voltage range.A given channel can be converted from one type to the other by post-translational modifications.²¹ A voltage-dependent phosphorylation was found to be an essential step for this switch,^22,23 although the kinase responsible for this conversion still needs to be uncovered.²⁴ In biophysical studies, mutant versions of the Arabidopsis K_weak channel subunit AKT2 have been created that showed impaired gating mode settings.^22,23 Recently, Gajdanowicz et al. generated transgenic Arabidopsis thaliana plants that express these mutant AKT2 channels in the background of the akt2-1 null-allele plant.²⁵ The major conclusion from analyses of these mutants is that the status switching of AKT2 from an inward-rectifying to a non-rectifying channel is crucial for plants to overcome energy-limiting conditions. This function of AKT2 could be correlated to its expression in phloem tissues. Selective expression of AKT2 under the control of the phloem companion cell-specific AtSUC2 promoter rescued the akt2-1 line, but conversely, selective expression of AKT2 under the control of the guard cell-specific GC1 promoter,²⁶ resulted in further impairment of plant growth (Fig. 1). By combining diverse experimental approaches with mathematical simulation methods, an existing model for phloem (re)loading^18,27 was fundamentally improved. This allowed the uncovering of a novel and interesting role of K⁺ in phloem physiology: K⁺ gradients present between the sieve element/companion cell (SE/CC) complex and the apoplast can serve as an energy source in phloem (re)loading processes. This “potassium battery” can be tapped by means of AKT2 regulation. This clarifies the observation of Deeken et al.²⁸ that in AKT2 loss-of-function mutant plants, assimilates leaking away from the sieve tube were not efficiently reloaded into the main phloem stream.Open in a separate windowFigure 1AKT2 expressed only in guard cells delays plant development. (A–C) Representative wild-type, akt2-1 and akt2-1+pGC1:AKT2 complementation plants grown for 7 weeks (A), 9 weeks (B) and 12 weeks (C) under 12-h day/12-h night conditions at normal light intensity (150 µmol m⁻² s⁻¹). (D) akt2-1+pGC1:AKT2 developed a similar number of leaves as the akt2-1 knock out plants, but bolting-time was delayed. (B and E) After 9 weeks, wild-type plants were at an advanced bolting stage, akt2-1 plants had started bolting, but only initial signs of bolting were visible in akt2-1+pGC1:AKT2 plants. (C and F) At 12 weeks, akt2-1 plants had caught up with the wild-type and akt2-1+pGC1:AKT2 was just starting to bolt, although rosette-leaves were showing clear signs of senescence. For the generation of akt2-1+pGC1:AKT2, the AKT2 cDNA was fused to the guard cell-specific GC1 promoter²⁶ kindly provided by J.I. Schroeder, San Diego. The pGC1:AKT2 construct was cloned into pGreen0229-35S by replacing the 35S promoter and then transformed into the akt2-1 knockout plant. All seeds were cold-treated for 24 h at 4°C. Plants were grown on artificial substrate (type GS-90, Einheitserde). After 2 weeks, seedlings were transferred to single pots. Plants were grown in 60% relative humidity at 21°C during the day and 18°C at night. Phenotypical analyses were done in the middle of the day. Data are shown as means ± SD of n ≥ 9 plants. Statistical analyses using Student''s t test: (D, WT/akt2-1: p < 2e-08; D, WT/pGC-AKT2: p < 2e-08; D, akt2-1/pGC-AKT2: p < 5e-03; E, WT/akt2-1: p < 4e-06; E, WT/pGC-AKT2: p < 1e-10; E, akt2-1/pGC-AKT2: p < 5e-04; F, WT/akt2-1: p = 0.51; F, WT/pGC-AKT2: p < 1e-10; F, akt2-1/pGC-AKT2: p < 1e-10).AKT2 expression is especially abundant in phloem tissues and the root stele, both of which are characterized by a poor availability of oxygen.^29,30 This local internal hypoxia impairs respiratory activity of the vascular tissue and concomitantly, respiratory ATP production is reduced.³¹ As a consequence, phloem transport is very susceptible to decreasing oxygen supply to the plant.^29,32 It is therefore comprehensible that the above mentioned support by the K⁺ driving force for sucrose retrieval is especially relevant in the phloem. Indeed Gajdanowicz et al.²⁵ showed that transgenic plants lacking the AKT2 K⁺ channel were severely impaired in growth when exposed to mild hypoxia (10% v:v), whereas growth of wild-type plants was unaffected by this treatment. These observations illustrate the importance of biochemical flexibility in plant cells to cope with the energetic consequences of the steep oxygen concentration gradients that generally occur in plant stems and roots.In fact, the role of K⁺ gradients in driving sugar, amino acid and organic acid transport across plant cell membranes was first suggested several decades ago.^33,34 Experimental evidence for this concept was provided by various tests in which pieces of plant tissue were incubated in solutions with different K⁺ concentrations and pH levels.^33,34 Unfortunately, at that time the lack of genetic information to support this hypothesis (e.g., identifying transporter proteins that could provide a molecular mechanism to explain the working mechanism of substrate transport driven by a K⁺-motive force) resulted in this idea falling into oblivion. Indeed, the unequivocal experimental observation of this new role of K⁺ gradients in phloem reloading is extremely challenging. Under normal experimental conditions, K⁺ fluxes and sucrose fluxes are coupled during phloem loading in source tissues and unloading in sink tissues. Nonetheless, computational simulations predict that under certain conditions, a local K⁺/Suc antiport is also thermodynamically possible. In this antiport system, the energy from the K⁺ gradient is used to transport Suc into the phloem. This process is only transient; flooding the apoplast with K⁺ will decrease the K⁺ gradient. However, the gradient can be maintained for longer if surrounding cells take up the apoplastic K⁺ for their own use. A K⁺/Suc antiport will not occur in obvious sink or source tissues since the energy balances in such cells are fundamentally different. Consequently, in these tissues only the coupled symport of K⁺ and Suc can be observed. However, the computational predictions allowed the identification of the experimental conditions under which the effect of the K⁺/Suc antiport system is empirically observable at the whole plant level.An essential role in the regulation of AKT2 is played by (de)phosphorylation events of serine residues at positions S210 and S329. The replacement of both serines by asparagine (AKT2-S210N-S329N) resulted in a K⁺-selective leak that is locked in a continuously open mode when the channels are expressed in Xenopus oocytes. Under certain conditions, plants expressing the AKT2-S210N-S329N mutation showed growth benefits over wild-type plants; akt2-1+AKT2-S210N-S329N plants reach the generative state faster, possess an increased number of leaves and increased fresh weight (Fig. 2). Intuitively, one would expect a continuously open channel to cause severe problems for the plant, not a benefit as was observed here. We therefore have to postulate that phosphorylation at residues AKT2-S210 and AKT2-S329 is insufficient for converting AKT2 from an inward-rectifying into a non-rectifying channel; other, as yet unknown mechanisms, must contribute to the switch in the AKT2 gating mode. Such a concept would correspond to results that would otherwise be hard to explain. For instance, when both serine residues were replaced by glutamate, the mutant AKT2-S210E-S329E still showed wild-type characteristics.²² The S to E substitution is expected to mimic the phosphorylated state better than the S to N replacement. Furthermore, position AKT2-K197 has a fundamental influence on the AKT2 gating mode.²³ AKT2 mutants with that particular lysine substituted with a serine are far less sensitive towards (de)phosphorylation; they display the characteristics of a pure inward-rectifying K⁺ channel,²³ and transgenic Arabidopsis plants expressing AKT2 channels with this substitution showed the characteristics of akt2-1 knock-out plants.²⁵ Initially, it was proposed that the positive charge is important for sensitizing AKT2 to phosphorylation. However, the charge-conserving mutant AKT2-K197R is similar to the charge inverting mutant AKT2-K197D,²³ a purely inward-rectifying channel (Fig. 3). We therefore need to take into account that in plants, K197 may also be a target of post-translational modification.³⁵ At present, we can explain the beneficial effect of the AKT2-S210N-S329N mutant on plant growth only by a multiple step regulation of AKT2 (Fig. 4). The double-N mutation would then bypass the phosphorylation step, but AKT2-S210N-S329N could still be deregulated into an inward-rectifying channel. Thus, AKT2 can be considered as a highly specialized K_in channel that can be converted into a leak-like channel by a cascade of post-translational modification steps.Open in a separate windowFigure 2Plants expressing the AKT2-S210N-S329N mutant reach the generative state faster than wild-type plants. The mutant channel AKT2-S210N-S329N was expressed under the control of the native AKT2 promoter in the akt2-1 knock-out background. (A) Photos of representative Arabidopsis thaliana plants grown 7 weeks under short day conditions (12-h day/12-h night, light intensity = 150 µE m⁻²s⁻¹). Seven weeks after sowing, plants expressing only AKT2-S210N-S329N mutant channels (n = 22) differed significantly (Student''s t test, p < 4e-05) from wild-type plants (n = 20) in the height of the main inflorescent stalk (B) and fresh weight (C). At later time points, these differences decrease.²⁵Open in a separate windowFigure 3The mutant AKT2-K197R channel is inward-rectifying. Steady-state current-voltage characteristics measured at the end of activation voltage steps. Currents were normalized to the current values measured at −145 mV in 10 mM K⁺ and are shown as means ± SD (n = 6).Open in a separate windowFigure 4Minimal model for AKT2 gating-mode regulation. To switch AKT2 from an inward-rectifying into a non-rectifying channel, at least two post-translational steps are postulated. (1) Phosphorylation at residues AKT2-S210 and AKT2-S329 (transitions [1]→[2] and [3]→[4]) and (2) a yet unknown modification that most likely involves the residue AKT2-K197 (transitions [1]→[3] and [2]→[4]). Only after both modifications will AKT2 allow the efflux of K⁺ (state [4]).