Effective half-lives for 137Cs in dairy milk from alpine ecosystems and the controlling factors.

Alpine regions in the federal state of Salzburg (Austria) have been intensively contaminated by Chernobyl fallout, necessitating long-term monitoring programs. The sites predominately affected are those in areas with soil developed on silicate bedrock, as these soils tend to be acidic, favouring high transfer factors for 137Cs. In addition, nutrient deficiency, low mineral and high organic matter content, and tough climatic conditions are causing the slow migration of 137Cs in the soil, which are associated with long effective half-lives in the biosphere. As a quantitative measure for effective half-lives, milk has been collected at nine alpine seasonal stock farming sites since 1988; at four sites, the monitoring is still ongoing (2020). For the period between 1999 and 2020, the decrease of 137Cs can be reasonably fitted with one effective half-life describing the time-trend. The effective half-lives obtained by this procedure vary between 9.3 ± 0.9 years and 18.8 ± 3.4 years. The effective half-lives show a weak negative correlation with the half-value depth of 137Cs, defined as the depth of the upper soil layer containing half of the deposited fallout inventory. The majority of the inventory is bound in the rooting zone of 0-10 cm, which is reflected by the small half value depths in the range between 3.2 and 4.4 cm. The soils investigated are acidic with pH values between 3.78 and 4.88, showing a pronounced negative correlation with the effective half-lives of 137Cs in milk. The data indicate that in these soils rich in organic matter, which are also almost totally devoid of clay minerals and have a very low clay size fraction, pH may be the dominating factor influencing the effective half-lives of 137Cs plant uptake and the subsequent contamination of milk.

Local Insect Availability Partly Explains Geographical Differences in Floral Visitor Assemblages of Arum maculatum L. (Araceae).

Geographical variation in abundance and composition of pollinator assemblages may result in variable selection pressures among plant populations and drive plant diversification. However, there is limited knowledge on whether differences in local visitor and pollinator assemblages are the result of site-specific strategies of plants to interact with their pollinators and/or merely reflect the pollinator availability at a given locality. To address this question, we compared locally available insect communities obtained by light-trapping with assemblages of floral visitors in populations of Arum maculatum (Araceae) from north vs. south of the Alps. We further investigated whether and how the abundance of different visitors affects plants' female reproductive success and examined the pollen loads of abundant visitors. Local insect availability explained inter-regional differences in total visitor abundance, but only partly the composition of visitor assemblages. Northern populations predominantly attracted females of Psychoda phalaenoides (Psychodidae, Diptera), reflecting the high availability of this moth fly in this region. More generalized visitor assemblages, including other psychodid and non-psychodid groups, were observed in the south, where the availability of P. phalaenoides/Psychodidae was limited. Fruit set was higher in the north than in the south but correlated positively in both regions with the abundance of total visitors and psychodids; in the north, however, this relationship disappeared when visitor abundances were too high. High pollen loads were recorded on both psychodids and other Diptera. We demonstrate for the first time that the quantitative assessment of floral visitor assemblages in relation to locally available insect communities is helpful to understand patterns of geographical variation in plant-pollinator interactions. This combined approach revealed that geographical differences in floral visitors of A. maculatum are only partly shaped by the local insect availability. Potential other factors that may contribute to the geographical pattern of visitor assemblages include the region-specific attractiveness of this plant species to flower visitors and the population-specific behavior of pollinators.

Antennae of psychodid and sphaerocerid flies respond to a high variety of floral scent compounds of deceptive Arum maculatum L.

Insect-pollinated plants often release complex mixtures of floral scents to attract their pollinators. Yet scent compounds eliciting physiological or behavioural responses in pollinators have only been identified in few plant species. The sapromyiophilous aroid Arum maculatum releases a highly diverse dung-like scent with overall more than 300 different compounds recorded so far to attract its psychodid and other fly pollinators. The volatiles' role in pollinator attraction is mostly unknown. To identify potential behaviourally active compounds, we recorded electroantennographic responses of four Psychodidae and one Sphaeroceridae species to (1) inflorescence scents of A. maculatum and (2) the scents released by cow dung, likely imitated by the plant species. Here we show that these flies are sensitive to 78 floral volatiles of various chemical classes, 18 of which were also found in cow dung. Our study, which for the first time determined physiologically active compounds in the antennae of Psychoda spp. and Sphaeroceridae, identified various volatiles not known to be biologically active in any floral visitors so far. The obtained results help deciphering the chemical basis that enables A. maculatum and other plants, pollinated by psychodids and sphaerocerids, to attract and deceive their pollinators.

Variation in scent amount but not in composition correlates with pollinator visits within populations of deceptive Arum maculatum L. (Araceae).

Floral scent is vital for pollinator attraction and varies among and within plant species. However, little is known about how inter-individual variation in floral scent affects the abundance and composition of floral visitor assemblages within populations. Moreover, for deceptive plants it is predicted that intra-population variation in scent can be maintained by negative frequency-dependent selection, but empirical evidence is still lacking. To investigate the ecological and evolutionary relations between inter-individual scent variation (i.e., total emission and composition) and floral visitors in deceptive plants, we studied floral scent, visitor assemblages, and fruit set in two populations of fly-pollinated (Psychodidae, Sphaeroceridae; Diptera) and deceptive Arum maculatum from Austria (JOS) and northern Italy (DAO). By correlating individual data on floral scent and visitor assemblages, we show that inter-individual variation in floral scent partly explains variation in visitor assemblages. The quantity of floral scent emitted per individual correlated positively with visitor abundance in both populations but explained visitor composition only in DAO, where strongly scented inflorescences attracted more sphaerocerid flies. However, in each population, the composition of floral scent did not correlate with the composition of floral visitors. There was also no evidence of negative frequency-dependent selection on floral scent. Instead, in JOS, more frequent scent phenotypes attracted more pollinators and were more likely to set an infructescence than rarer ones. Our results show that floral scent, despite being key in pollinator attraction in A. maculatum, only partly explains variation in pollinator abundance and composition. Overall, this study is the first to shed light on the importance of inter-individual variation in floral scent in explaining floral visitor assemblages at the population level in a deceptive plant species.

Floral Scents of a Deceptive Plant Are Hyperdiverse and Under Population-Specific Phenotypic Selection.

Floral scent is a key mediator in plant-pollinator interactions. However, little is known to what extent intraspecific scent variation is shaped by phenotypic selection, with no information yet in deceptive plants. In this study, we collected inflorescence scent and fruit set of the deceptive moth fly-pollinated Arum maculatum L. (Araceae) from six populations north vs. five populations south of the Alps, accumulating to 233 samples in total, and tested for differences in scent, fruit set, and phenotypic selection on scent across this geographic barrier. We recorded 289 scent compounds, the highest number so far reported in a single plant species. Most of the compounds occurred both north and south of the Alps; however, plants of the different regions emitted different absolute and relative amounts of scent. Fruit set was higher north than south of the Alps, and some, but not all differences in scent could be explained by differential phenotypic selection in northern vs. southern populations. This study is the first to provide evidence that floral scents of a deceptive plant are under phenotypic selection and that phenotypic selection is involved in shaping geographic patterns of floral scent in such plants. The hyperdiverse scent of A. maculatum might result from the imitation of various brood substrates of its pollinators.

Extracellular proteolytic cascade in tomato activates immune protease Rcr3.

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.

The de Novo Reference Genome and Transcriptome Assemblies of the Wild Tomato Species Solanum chilense Highlights Birth and Death of NLR Genes Between Tomato Species.

Wild tomato species, like Solanum chilense, are important germplasm resources for enhanced biotic and abiotic stress resistance in tomato breeding. S. chilense also serves as a model to study adaptation of plants to drought and the evolution of seed banks. The absence of a well-annotated reference genome in this compulsory outcrossing, very diverse species limits in-depth studies on the genes involved.We generated ∼134 Gb of DNA and 157 Gb of RNA sequence data for S chilense, which yielded a draft genome with an estimated length of 914 Mb, encoding 25,885 high-confidence predicted gene models, which show homology to known protein-coding genes of other tomato species. Approximately 71% of these gene models are supported by RNA-seq data derived from leaf tissue samples. Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis of predicted gene models retrieved 93.3% of BUSCO genes. To further verify the genome annotation completeness and accuracy, we manually inspected the NLR resistance gene family and assessed its assembly quality. We find subfamilies of NLRs unique to S. chilense Synteny analysis suggests significant degree of the gene order conservation between the S. chilense, S. lycopersicum and S. pennellii genomesWe generated the first genome and transcriptome sequence assemblies for the wild tomato species Solanum chilense and demonstrated their value in comparative genomics analyses. These data are an important resource for studies on adaptation to biotic and abiotic stress in Solanaceae, on evolution of self-incompatibility and for tomato breeding.

Screen of Non-annotated Small Secreted Proteins of Pseudomonas syringae Reveals a Virulence Factor That Inhibits Tomato Immune Proteases.

Pseudomonas syringae pv. tomato DC3000 (PtoDC3000) is an extracellular model plant pathogen, yet its potential to produce secreted effectors that manipulate the apoplast has been under investigated. Here we identified 131 candidate small, secreted, non-annotated proteins from the PtoDC3000 genome, most of which are common to Pseudomonas species and potentially expressed during apoplastic colonization. We produced 43 of these proteins through a custom-made gateway-compatible expression system for extracellular bacterial proteins, and screened them for their ability to inhibit the secreted immune protease C14 of tomato using competitive activity-based protein profiling. This screen revealed C14-inhibiting protein-1 (Cip1), which contains motifs of the chagasin-like protease inhibitors. Cip1 mutants are less virulent on tomato, demonstrating the importance of this effector in apoplastic immunity. Cip1 also inhibits immune protease Pip1, which is known to suppress PtoDC3000 infection, but has a lower affinity for its close homolog Rcr3, explaining why this protein is not recognized in tomato plants carrying the Cf-2 resistance gene, which uses Rcr3 as a co-receptor to detect pathogen-derived protease inhibitors. Thus, this approach uncovered a protease inhibitor of P. syringae, indicating that also P. syringae secretes effectors that selectively target apoplastic host proteases of tomato, similar to tomato pathogenic fungi, oomycetes and nematodes.

Functional Divergence of Two Secreted Immune Proteases of Tomato.

Rcr3 and Pip1 are paralogous secreted papain-like proteases of tomato. Both proteases are inhibited by Avr2 from the fungal pathogen Cladosporium fulvum, but only Rcr3 acts as a co-receptor for Avr2 recognition by the tomato Cf-2 immune receptor. Here, we show that Pip1-depleted tomato plants are hyper-susceptible to fungal, bacterial, and oomycete plant pathogens, demonstrating that Pip1 is an important broad-range immune protease. By contrast, in the absence of Cf-2, Rcr3 depletion does not affect fungal and bacterial infection levels but causes increased susceptibility only to the oomycete pathogen Phytophthora infestans. Rcr3 and Pip1 reside on a genetic locus that evolved over 36 million years ago. These proteins differ in surface-exposed residues outside the substrate-binding groove, and Pip1 is 5- to 10-fold more abundant than Rcr3. We propose a model in which Rcr3 and Pip1 diverged functionally upon gene duplication, possibly driven by an arms race with pathogen-derived inhibitors or by coevolution with the Cf-2 immune receptor detecting inhibitors of Rcr3, but not of Pip1.

PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R.  solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

The current status of the elemental defense hypothesis in relation to pathogens.

Metal hyperaccumulating plants are able to accumulate exceptionally high concentrations of metals, such as zinc, nickel, or cadmium, in their aerial tissues. These metals reach concentrations that would be toxic to most other plant species. This trait has evolved multiple times independently in the plant kingdom. Recent studies have provided new insight into the ecological and evolutionary significance of this trait, by showing that some metal hyperaccumulating plants can use high concentrations of accumulated metals to defend themselves against attack by pathogenic microorganisms and herbivores. Here, we review the evidence that metal hyperaccumulation acts as a defensive trait in plants, with particular emphasis on plant-pathogen interactions. We discuss the mechanisms by which defense against pathogens might have driven the evolution of metal hyperaccumulation, including the interaction of this trait with other forms of defense. In particular, we consider how physiological adaptations and fitness costs associated with metal hyperaccumulation could have resulted in trade-offs between metal hyperaccumulation and other defenses. Drawing on current understanding of the population ecology of metal hyperaccumulator plants, we consider the conditions that might have been necessary for metal hyperaccumulation to be selected as a defensive trait, and discuss the likelihood that these were fulfilled. Based on these conditions, we propose a possible scenario for the evolution of metal hyperaccumulation, in which selective pressure for resistance to pathogens or herbivores, combined with gene flow from non-metallicolous populations, increases the likelihood that the metal hyperaccumulating trait becomes established in plant populations.

The structural basis of specific protease-inhibitor interactions at the plant-pathogen interface.

Antagonistic host-pathogen interactions offer intriguing insights into coevolutionary processes at the molecular level. Studies on secreted immune proteases from the model plant tomato and their interactions with different unrelated pathogen-derived inhibitors revealed that the inhibitors exhibit a remarkable selectivity towards different host proteases, and that the host proteases accumulate variant residues at the interaction surfaces that interfere with inhibitor binding. Here, we summarize and discuss the recent findings and use structural models to identify the molecular features underpinning protease selectivity. The observed basic principles translate to other examples of secreted immune hydrolases and their putative inhibitors.

Balancing selection at the tomato RCR3 Guardee gene family maintains variation in strength of pathogen defense.

Coevolution between hosts and pathogens is thought to occur between interacting molecules of both species. This results in the maintenance of genetic diversity at pathogen antigens (or so-called effectors) and host resistance genes such as the major histocompatibility complex (MHC) in mammals or resistance (R) genes in plants. In plant-pathogen interactions, the current paradigm posits that a specific defense response is activated upon recognition of pathogen effectors via interaction with their corresponding R proteins. According to the "Guard-Hypothesis," R proteins (the "guards") can sense modification of target molecules in the host (the "guardees") by pathogen effectors and subsequently trigger the defense response. Multiple studies have reported high genetic diversity at R genes maintained by balancing selection. In contrast, little is known about the evolutionary mechanisms shaping the guardee, which may be subject to contrasting evolutionary forces. Here we show that the evolution of the guardee RCR3 is characterized by gene duplication, frequent gene conversion, and balancing selection in the wild tomato species Solanum peruvianum. Investigating the functional characteristics of 54 natural variants through in vitro and in planta assays, we detected differences in recognition of the pathogen effector through interaction with the guardee, as well as substantial variation in the strength of the defense response. This variation is maintained by balancing selection at each copy of the RCR3 gene. Our analyses pinpoint three amino acid polymorphisms with key functional consequences for the coevolution between the guardee (RCR3) and its guard (Cf-2). We conclude that, in addition to coevolution at the "guardee-effector" interface for pathogen recognition, natural selection acts on the "guard-guardee" interface. Guardee evolution may be governed by a counterbalance between improved activation in the presence and prevention of auto-immune responses in the absence of the corresponding pathogen.

A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf) during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

Targets of selection in a disease resistance network in wild tomatoes.

Studies combining comparative genomics and information on biochemical pathways have revealed that protein evolution can be affected by the amount of pleiotropy associated with a particular gene. The amount of pleiotropy, in turn, can be a function of the position at which a gene operates in a pathway and the pathway structure. Genes that serve as convergence points and have several partners (so-called hubs) often show the greatest constraint and hence the slowest rate of protein evolution. In this article, we have studied five genes (Pto, Fen, Rin4, Prf and Pfi) in a defence signalling network in a wild tomato species, Solanum peruvianum. These proteins operate together and contribute to bacterial resistance in tomato. We predicted that Prf (and possibly Pfi), which serves as a convergence point for upstream signals, should show greater evolutionary constraint. However, we found instead that two of the genes which potentially interact with pathogen ligands, Rin4 and Fen, have evolved under strong evolutionary constraint, whereas Prf and Pfi, which probably function further downstream in the network, show evidence of balancing selection. This counterintuitive observation may be probable in pathogen defence networks, because pathogens may target positions throughout resistance networks to manipulate or nullify host resistance, thereby leaving a molecular signature of host-parasite co-evolution throughout a single network.