Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

To control net sodium (Na+) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na+/H+ antiporter SOS1 to achieve Na+ efflux at the root and Na+ loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na+ unloading off the xylem. Together, these opposing transport systems govern the partition of Na+ within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na+ fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na+ export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na+ transport systems operating in vascular plants controlling plant tolerance to salinity.

S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

A large-scale dataset reveals taxonomic and functional specificities of wild bee communities in urban habitats of Western Europe.

Wild bees are declining, mainly due to the expansion of urban habitats that have led to land-use changes. Effects of urbanization on wild bee communities are still unclear, as shown by contrasting reports on their species and functional diversities in urban habitats. To address this current controversy, we built a large dataset, merging 16 surveys carried out in 3 countries of Western Europe during the past decades, and tested whether urbanization influences local wild bee taxonomic and functional community composition. These surveys encompassed a range of urbanization levels, that were quantified using two complementary metrics: the proportion of impervious surfaces and the human population density. Urban expansion, when measured as a proportion of impervious surfaces, but not as human population density, was significantly and negatively correlated with wild bee community species richness. Taxonomic dissimilarity of the bee community was independent of both urbanization metrics. However, occurrence rates of functional traits revealed significant differences between lightly and highly urbanized communities, for both urbanization metrics. With higher human population density, probabilities of occurrence of above-ground nesters, generalist and small species increased. With higher soil sealing, probabilities of occurrence of above-ground nesters, generalists and social bees increased as well. Overall, these results, based on a large European dataset, suggest that urbanization can have negative impacts on wild bee diversity. They further identify some traits favored in urban environments, showing that several wild bee species can thrive in cities.

Phylogenetic, functional and taxonomic responses of wild bee communities along urbanisation gradients.

Increasing urbanisation is one of the primary drivers of land-use change that threaten biodiversity. Wild bee communities have been reported with contrasting responses to urbanisation, with varying effects on abundance and taxonomical diversity. The suite of functional traits exhibited by wild bee species might determine their persistence in urban areas. Urbanisation thus can impose an environmental filter with potential consequences on the functional and phylogenetical diversity of wild bee communities. Here, we sampled 2944 wild bee specimens from 156 species in 29 sites located along an urbanisation gradient using a replicated design in three mid-sized cities in the Loire valley (France). We show that urban landscape cover has a negative effect on overall species richness and taxonomical diversity indices, while total abundance remains constant. Species loss was taxon dependent, mainly driven by Andrenidae and Halictidae. Only a few species, especially of the genus Lasioglossum, were positively affected by the urban landscape cover. Urban and peri-urban areas differed in their composition of bee assemblages. Species turnover was the main component of beta diversity, driving community dissimilarities through the urban gradient. Urbanisation favours bees with small body sizes, social structure and extended flight periods but did not affect the phylogenetic or the functional diversity of communities. Our findings have implications for understanding the factors involved in the environmental filter exerted through the urban gradient on bee communities helping to implement conservation measures and managing urban spaces for bees.

Distinct Roles of N-Terminal Fatty Acid Acylation of the Salinity-Sensor Protein SOS3.

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N-myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S-acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis. These results are evidence of a novel S-acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S-acylation.

A DNA barcode-based survey of wild urban bees in the Loire Valley, France.

The current decline of wild bees puts important ecosystem services such as pollination at risk. Both inventory and monitoring programs are needed to understand the causes of wild bee decline. Effective insect monitoring relies on both mass-trapping methods coupled with rapid and accurate identifications. Identifying wild bees using only morphology can be challenging, in particular, specimens from mass-trapped samples which are often in poor condition. We generated DNA barcodes for 2931 specimens representing 157 species (156 named and one unnamed species) and 28 genera. Automated cluster delineation reveals 172 BINs (Barcodes Index Numbers). A total of 36 species (22.93%) were found in highly urbanized areas. The majority of specimens, representing 96.17% of the species barcoded form reciprocally exclusive groups, allowing their unambiguous identification. This includes several closely related species notoriously difficult to identify. A total of 137 species (87.26%) show a "one-to-one" match between a named species and the BIN assignment. Fourteen species (8.92%) show deep conspecific lineages with no apparent morphological differentiation. Only two species pairs shared the same BIN making their identification with DNA barcodes alone uncertain. Therefore, our DNA barcoding reference library allows reliable identification by non-experts for the vast majority of wild bee species in the Loire Valley.

Does social thermal regulation constrain individual thermal tolerance in an ant species?

In ants, social thermal regulation is the collective maintenance of a nest temperature that is optimal for individual colony members. In the thermophilic ant Aphaenogaster iberica, two key behaviours regulate nest temperature: seasonal nest relocation and variable nest depth. Outside the nest, foragers must adapt their activity to avoid temperatures that exceed their thermal limits. It has been suggested that social thermal regulation constrains physiological and morphological thermal adaptations at the individual level. We tested this hypothesis by examining the foraging rhythms of six populations of A. iberica, which were found at different elevations (from 100 to 2,000 m) in the Sierra Nevada mountain range of southern Spain. We tested the thermal resistance of individuals from these populations under controlled conditions. Janzen's climatic variability hypothesis (CVH) states that greater climatic variability should select for organisms with broader temperature tolerances. We found that the A. iberica population at 1,300 m experienced the most extreme temperatures and that ants from this population had the highest heat tolerance (LT50 = 57.55°C). These results support CVH's validity at microclimatic scales, such as the one represented by the elevational gradient in this study. Aphaenogaster iberica maintains colony food intake levels across different elevations and mean daily temperatures by shifting its rhythm of activity. This efficient colony-level thermal regulation and the significant differences in individual heat tolerance that we observed among the populations suggest that behaviourally controlled thermal regulation does not constrain individual physiological adaptations for coping with extreme temperatures.

En hormigas, la termorregulación social es el mantenimiento colectivo de la temperatura del nido óptima para los individuos de la colonia. En la hormiga termófila Aphaenogaster iberica, hay dos comportamientos clave que regulan la temperatura del nido: la reubicación estacional y la profundidad variable del nido. Fuera del nido, las obreras recolectoras deben adaptar su actividad para evitar las temperaturas que excedan sus límites térmicos. Se ha sugerido que la termorregulación social limita las adaptaciones térmicas a nivel individual, fisiológicas y morfológicas. Examinamos esta hipótesis, estudiando los ritmos de actividad de recolección de alimento en seis poblaciones de A. iberica, a distinta altitud, desde 100 m a 2,000 m, en las montañas de Sierra Nevada, en el sur de España. Y analizamos la resistencia térmica de los individuos de estas poblaciones, en condiciones controladas. La Hipótesis de la Variabilidad Climática de Janzen (CVH) postula que una mayor variabilidad climática selecciona organismos con tolerancias térmicas más amplias. Encontramos que la población de 1,300 m era la que presentaba la mayor variabilidad climática, y que las hormigas de esta población tiene la mayor resistencia térmica individual (LT50 = 57.55°C), lo que confirma la validez de la CVH a una escala microclimática en el gradiente altitudinal estudiado. Encontramos que A. iberica puede compensar por la disminución de la temperatura media que acompaña al incremento en elevación. Las hormigas pueden cambiar sus ritmos de actividad sin afectar la entrada de alimento en la colonia, que tampoco se ve afectada por la elevación o la temperatura media diaria. A pesar de esta eficiente termorregulación a nivel de colonia, las diferencias estadísticamente significativas entre poblaciones observadas en la tolerancia térmica individual sugieren que la termorregulación controlada comportalmente no limita las adaptaciones fisiológicas individuales para enfrentarse a temperaturas extremas.

A Critical Role of Sodium Flux via the Plasma Membrane Na+/H+ Exchanger SOS1 in the Salt Tolerance of Rice.

Rice (Oryza sativa) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na+) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na+/H+ exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na+ efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na+ flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na+ intake and impaired Na+ loading into the xylem, thus indicating that SOS1 controls net root Na+ uptake and long-distance Na+ transport to shoots. The acute Na+ sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1 In summary, the plasma membrane Na+/H+ exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na+ homeostasis and possibly contributing to the sensing of sodicity stress.

K+ Efflux Antiporters 4, 5, and 6 Mediate pH and K+ Homeostasis in Endomembrane Compartments.

KEA4, KEA5, and KEA6 are members of the Arabidopsis (Arabidopsis thaliana) K+ efflux antiporter (KEA) family that share high sequence similarity but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. KEA4, KEA5, and KEA6 had similar tissue expression patterns, and the three KEA proteins localized to the Golgi, the trans-Golgi network, and the prevacuolar compartment/multivesicular bodies, suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant kea4 kea5 kea6 had small rosettes, short seedlings, and was sensitive to low K+ availability and to the sodicity imposed by high salinity. Also, the kea4 kea5 kea6 mutant plants had a reduced luminal pH in the Golgi, trans-Golgi network, prevacuolar compartment, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, and KEA6 as well as endosomal Na+/H+exchanger5 (NHX5) and NHX6 acted coordinately to facilitate endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters NHX1 and NHX2 in the kea4 kea5 kea6 mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the kea4 kea5 kea6 mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K+ transporters functioning in maintaining pH and ion homeostasis in the endomembrane network.

Queen Control or Queen Signal in Ants: What Remains of the Controversy 25 Years After Keller and Nonacs' Seminal Paper?

Ant queen pheromones (QPs) have long been known to affect colony functioning. In many species, QPs affect important reproductive functions such as diploid larvae sexualization and egg-laying by workers, unmated queens (gynes), or other queens. Until the 1990s, these effects were generally viewed to be the result of queen manipulation through the use of coercive or dishonest signals. However, in their seminal 1993 paper, Keller and Nonacs challenged this idea, suggesting that QPs had evolved as honest signals that informed workers and other colony members of the queen's presence and reproductive state. This paper has greatly influenced the study of ant QPs and inspired numerous attempts to identify fertility-related compounds and test their physiological and behavioral effects. In the present article, we review the literature on ant QPs in various contexts and pay special attention to the role of cuticular hydrocarbons (CHCs). Although the controversy generated by Keller and Nonacs' (Anim Behav 45:787-794, 1993) paper is currently less intensively debated, there is still no clear evidence which allows the rejection of the queen control hypothesis in favor of the queen signal hypothesis. We argue that important questions remain regarding the mode of action of QPs, and their targets which may help understanding their evolution.

Origin and distribution of desert ants across the Gibraltar Straits.

The creation of geographic barriers has long been suspected to contribute to the formation of new species. We investigated the phylogeography of desert ants in the western Mediterranean basin in order to elucidate their mode of diversification. These insects which have a low dispersal capacity are recently becoming important model systems in evolutionary studies. We conducted an extensive sampling of species belonging to the Cataglyphis albicans group in the Iberian Peninsula (IP) and the northern Morocco (North Africa; NA). We then combined genetic, chemical and morphological analyses. The results suggest the existence of at least three and five clades in the IP and NA, respectively, whose delineation partially encompass current taxonomic classification. The three Iberian clades are monophyletic, but their origin in NA is uncertain (79% and 22% for Bayesian and Maximum Likelihood support, respectively). The estimation of divergence time suggests that a speciation process was initiated after the last reopening of the Gibraltar Straits ≈5.33 Ma. In the IP, the clades are parapatric and their formation may have been triggered by the fragmentation of a large population during the Pleistocene due to extended periods of glaciation. This scenario is supported by demographic analyses pointing at a recent expansion of Iberian populations that contrasts with the progressive contraction of the NA clades. Niche modeling reveals that this area, governed by favorable climatic conditions for desert ants, has recently increased in the IP and decreased in NA. Altogether, our data points at geoclimatic events as major determinants of species formation in desert ants, reinforcing the role of allopatric speciation.

Social coercion of larval development in an ant species.

Ants provide one of the best examples of the division of labor in animal societies. While the queens reproduce, workers generally refrain from laying eggs and dedicate themselves exclusively to domestic tasks. In many species, the small diploid larvae are bipotent and can develop either into workers or queens depending mostly on environmental cues. This generates a conflicting situation between the adults that tend to rear a majority of larvae into workers and the larvae whose individual interest may be to develop into reproductive queens. We tested the social regulation of larval caste fate in the fission-performing ant Aphaenogaster senilis. We first observed interactions between resident workers and queen- and worker-destined larvae in presence/absence of the queen. The results show that workers tend to specifically eliminate queen-destined larvae when the queen is present but not when she is absent or imprisoned in a small cage allowing for volatile pheromone exchanges. In addition, we found that the presence of already developed queen-destined larvae does not inhibit the development of younger still bipotent larvae into queens. Finally, we analyzed the cuticular hydrocarbon profiles of queen- and worker-destined larvae and found no significant quantitative or qualitative difference. Interestingly, the total amount of hydrocarbons on both larval castes is extremely low, which lends credence on the chemical insignificance hypothesis of larval ants. Overall, our results suggest that workers control larval development and police larvae that would develop into queens instead of workers. Such policing behavior is similar in many aspects to what is known of worker policing among adults.

The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+ /K+ homeostasis.

The location of major quantitative trait loci (QTL) contributing to stem and leaf [Na(+) ] and [K(+) ] was previously reported in chromosome 7 using two connected populations of recombinant inbred lines (RILs) of tomato. HKT1;1 and HKT1;2, two tomato Na(+) -selective class I-HKT transporters, were found to be closely linked, where the maximum logarithm of odds (LOD) score for these QTLs located. When a chromosome 7 linkage map based on 278 single-nucleotide polymorphisms (SNPs) was used, the maximum LOD score position was only 35 kb from HKT1;1 and HKT1;2. Their expression patterns and phenotypic effects were further investigated in two near-isogenic lines (NILs): 157-14 (double homozygote for the cheesmaniae alleles) and 157-17 (double homozygote for the lycopersicum alleles). The expression pattern for the HKT1;1 and HKT1;2 alleles was complex, possibly because of differences in their promoter sequences. High salinity had very little effect on root dry and fresh weight and consequently on the plant dry weight of NIL 157-14 in comparison with 157-17. A significant difference between NILs was also found for [K(+) ] and the [Na(+) ]/[K(+) ] ratio in leaf and stem but not for [Na(+) ] arising a disagreement with the corresponding RIL population. Their association with leaf [Na(+) ] and salt tolerance in tomato is also discussed.

Structural insights on the plant salt-overly-sensitive 1 (SOS1) Na(+)/H(+) antiporter.

The Arabidopsisthaliana Na(+)/H(+) antiporter salt-overly-sensitive 1 (SOS1) is essential to maintain low intracellular levels of toxic Na(+) under salt stress. Available data show that the plant SOS2 protein kinase and its interacting activator, the SOS3 calcium-binding protein, function together in decoding calcium signals elicited by salt stress and regulating the phosphorylation state and the activity of SOS1. Molecular genetic studies have shown that the activation implies a domain reorganization of the antiporter cytosolic moiety, indicating that there is a clear relationship between function and molecular structure of the antiporter. To provide information on this issue, we have carried out in vivo and in vitro studies on the oligomerization state of SOS1. In addition, we have performed electron microscopy and single-particle reconstruction of negatively stained full-length and active SOS1. Our studies show that the protein is a homodimer that contains a membrane domain similar to that found in other antiporters of the family and an elongated, large, and structured cytosolic domain. Both the transmembrane (TM) and cytosolic moieties contribute to the dimerization of the antiporter. The close contacts between the TM and the cytosolic domains provide a link between regulation and transport activity of the antiporter.

Activation of the plasma membrane Na/H antiporter Salt-Overly-Sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain.

The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex up-regulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.