Decoupled post-glacial history in mutualistic plant-insect interactions: insights from the yellow loosestrife (Lysimachia vulgaris) and its associated oil-collecting bees (Macropis europaea and M-fulvipes)

AimWe take a comparative phylogeographical approach to assess whether three species involved in a specialized oil-rewarding pollination system (i.e. Lysimachia vulgaris and two oil-collecting bees within the genus Macropis) show congruent phylogeographical trajectories during post-glacial colonization processes. Our working hypothesis is that within specialized mutualistic interactions, where each species relies on the co-occurrence of the other for survival and/or reproduction, partners are expected to show congruent evolutionary trajectories, because they are likely to have followed parallel migration routes and to have shared glacial refugia. LocationWestern Palaearctic. MethodsOur analysis relies on the extensive sampling of 104 Western Palaearctic populations (totalling 434, 159 and 74 specimens of Lysimachiavulgaris, Macropiseuropaea and Macropisfulvipes, respectively), genotyped with amplified fragment length polymorphism. Based on this, we evaluated the regional genetic diversity (Shannon diversity and allele rarity index) and genetic structure (assessed using structure, population networks, isolation-by-distance and spatial autocorrelation metrics) of each species. Finally, we compared the general phylogeographical patterns obtained. ResultsContrary to our expectations, the analyses revealed phylogeographical signals suggesting that the investigated organisms demonstrate independent post-glacial trajectories as well as distinct contemporaneous demographic parameters, despite their mutualistic interaction. Main conclusionsThe mutualistic partners investigated here are likely to be experiencing distinct and independent evolutionary dynamics because of their contrasting life-history traits (e.g. dispersal abilities), as well as distinct hubs and migration routes. Such conditions would prevent and/or erase any signature of co-structuring of lineages in space and time. As a result, the lack of phylogeographical congruence driven by differences in life-history traits might have arisen irrespective of the three species having shared similar Pleistocene glacial refugia.

Tracking the ghost of coevolution in specific plant-insect interactions : the case of the pollination system between the globeflower Trollius europaeus and seven european Chiastocheta flies

This study aims at understanding the evolutionary processes at work in specialized species interactions. Prom the macroevolutionary perspective, coevolution among specialized taxa was proposed to be one of the major processes generating biodiversity. We challenge this idea from the theoretical and practical perspective and through a literature review and show that the major hypotheses linking coevolutionary process with macroevolutionary patterns do not necessarily predict lineage co diversification and parallel speciation, limit¬ing the utility of the comparative phylogenenetic approach for investigating coevolution¬ary processes. We also point to the rarity of observed long-term coevolutionary dynamics among lineages and propose that coevolution rather occurs in shorter timescales, followed by ecological fitting. Prom the empirical point, we focus on the nursery pollination interaction between the European globeflower Trollius europaeus (Ranunculaceae) and its associated Chiastocheta flies (Anthomyiidae; Diptera) as a model system of evolution and maintenance of special¬ized interactions. The flies are obligate parasites of the seeds, but also pollinate the plant - it was thus proposed that both species are mutually dependent. Contrasting with the paradigm used for two decades of research on this system, we show that the female fitness component of the plant is similar in the populations with and without Chiastocheta. The plant is thus not exclusively dependent on the flies for reproduction. We discuss this result in the context of the factors responsible for the evolution of mutualistic systems. Understanding the evolution of a biological system requires understanding of its phylo- genetic context. Previous studies showed large mismatch between mtDNA phylogeny and morphological taxonomy in Chiastocheta. By using a large set of RAD-sequencing loci, we delineate the species limits that are congruent with morphology, and show that the discordance is best explained by the scenario of mitochondrial capture among fly species. Finally, we examine this system from a phylogeographic perspective, and identify the lack of congruence in spatial genetic structures of the plant and associated insects across their whole geographic range. The flies show lower numbers of spatial genetic groups than the plant, indicating that not all of the plant réfugia were shared by all the fly species or that the migration dynamics homogenized some of the groups. The incongruence in spatial genetic patterns indicates that fly migrations were largely independent from the genetic background of the plant, following rather a scenario of resource tracking, without the signature of coevolutionary process at this scale. Indeed, while the flies require the plant to survive climatic oscillations, the opposite is not true. Eventually, we show that there is no phylogenetic signal of spatial genetic structures, meaning that neither histories nor life- history traits are shared among closely related species and that species are characterized by unique trajectories of their genes. -- Cette étude vise à comprendre les processus évolutifs à l'oeuvre au sein d'interactions en¬tre espèces spécialisées. Du point de vue macroévolutif, la coévolution entre les taxons spécialisée a été considérée comme l'un des principaux processus générateur de biodiversité. Nous contestons cette idée du point de vue théorique et pratique à travers une revue de la littérature. Nous montrons que les hypothèses majeures reliant les processus coévolutifs avec les patterns de diversité au niveau macroévolutif ne prédisent pas nécessairement la co- diversification des lignées et leur spéciation parallèle, ce qui limite l'utilité de l'approche de phylogénie comparative pour étudier les processus coévolutifs . Nous rappelons également le peu d'exemples de dynamique coévolutive à long terme et proposons que la coévolution se produit plutôt dans des intervalles courts, suivis d'ajustements écologiques. Du point empirique, nous nous concentrons sur l'interaction de pollinisation entre le Trolle d'Europe Trollius europaeus (Ranunculaceae) et ses pollinisateurs associés, du genre Chiastocheta (Anthomyiidae; Diptera) en tant que système-modèle pour étudier l'évolution et le maintien des interactions spécialisées. Les mouches sont des parasites obligatoires des semences, mais pollinisent également la plante. Il a donc été proposé que les deux espèces soient mutuellement dépendantes. Contrastant avec le paradigme utilisé pendant deux décennies de recherche sur ce système, nous montrons, que la composante de fitness femelle de la plante est similaire dans les populations avec et sans Chiastocheta. La plante ne dépend donc pas exclusivement de son interaction avec les mouches pour la reproduction. Nous discutons de ce résultat dans le contexte des facteurs responsables de l'évolution des systèmes mutualistes. Comprendre l'évolution d'un système biologique nécessite la compréhension de son con- texte phylogénétique. Des études antérieures ont montré, chez Chiastocheta, de grandes disparités entre les phylogénies obtenues à partir d'ADN mitochondrial et la taxonomie basée sur les critères morphologiques. En utilisant un grand nombre de loci obtenus par RAD-sequencing, nous traçons les limites des espèces, qui concordent avec les car¬actéristiques morphologies, et montrons que la discordance s'explique en fait par un scénario de capture mitochondriale entre espèces de mouches. Enfin, nous examinons le système d'un point de vue phylogéographique, et identi¬fions les incohérences entre structurations génétiques spatiales de la plante et des insectes associés dans toute leur aire de distribution géographique. Les mouches présentent un nombre de groupes génétiques inférieur à la plante, indiquant que tous les refuges de la plante n'étaient pas partagés par toutes les espèces de mouches ou que les dynamiques migratoires ont homogénéisés certains des groupes chez les mouches. Les différences ob¬servées dans les patrons de structuration génétique spatiale indique que les migrations et dispersions des mouches ont été indépendantes du contexte génétique de la plante, et ces dernières ont été uniquement tributaires de la disponibilité des ressources, sans qu'il n'y ait de signature du processus de coévolution à cette échelle. En effet, tandis que les mouches ont besoin de la plante pour survivre aux oscillations climatiques, le contraire n'est pas exact. Finalement, nous montrons qu'il n'y a pas de signal phylogénétique des structurations génétiques spatiales chez les mouches, ce qui signifie que ni l'histoire, ni les traits d'histoire de vie ne sont partagés entre les espèces phylogénétiquement proches et que les espèces sont caractérisées par des trajectoires uniques de leurs gènes.

Plant-Herbivore Interaction in a Fragmented Landscape: Local Adaptation and Inbreeding

Reciprocal selection between interacting species is a major driver of biodiversity at both the genetic and the species level. This reciprocal selection, or coevolution, has led to the diversification of two highly diverse and abundant groups of organisms, flowering plants and their insect herbivores. In heterogeneous environments, the outcome of coevolved species interactions is influenced by the surrounding community and/or the abiotic environment. The process of adaptation allows species to adapt to their local conditions and to local populations of interacting species. However, adaptation can be disrupted or slowed down by an absence of genetic variation or by increased inbreeding, together with the following inbreeding depression, both of which are common in small and isolated populations that occur in fragmented environments. I studied the interaction between a long-lived plant Vincetoxicum hirundinaria and its specialist herbivore Abrostola asclepiadis in the southwestern archipelago of Finland. I focused on mutual local adaptation of plants and herbivores, which is a demonstration of reciprocal selection between species, a prerequisite for coevolution. I then proceeded to investigate the processes that could potentially hamper local adaptation, or species interaction in general, when the population size is small. I did this by examining how inbreeding of both plants and herbivores affects traits that are important for interaction, as well as among-population variation in the effects of inbreeding. In addition to bi-parental inbreeding, in plants inbreeding can arise from self-fertilization which has important implications for mating system evolution. I found that local adaptation of the plant to its herbivores varied among populations. Local adaptation of the herbivore varied among populations and years, being weaker in populations that were most connected. Inbreeding caused inbreeding depression in both plants and herbivores. In some populations inbreeding depression in herbivore biomass was stronger in herbivores feeding on inbred plants than in those feeding on outbred ones. For plants it was the other way around: inbreeding depression in anti-herbivore resistance decreased when the herbivores were inbred. Underlying some of the among-population variation in the effects of inbreeding is variation in plant phenolic compounds. However, variation in the modification of phenolic compounds in the digestive tract of the herbivore did not explain the inbreeding depression in herbivore biomass. Finally, adult herbivores had a preference for outbred host plants for egg deposition, and herbivore inbreeding had a positive effect on egg survival when the eggs were exposed to predators and parasitoids. These results suggest that plants and herbivores indeed exert reciprocal selection, as demonstrated by the significant local adaptation of V. hirundinaria and A. asclepiadis to one another. The most significant cause of disruption of the local adaptation of herbivore populations was population connectivity, and thus probably gene flow. In plants local adaptation tended to increase with increasing genetic variation. Whether or not inbreeding depression occurred varied according to the life-history stage of the herbivore and/or the plant trait in question. In addition, the effects of inbreeding strongly depended on the population. Taken together, inbreeding modified plant-herbivore interactions at several different levels, and can thus affect the strength of reciprocal selection between species. Thus inbreeding has the potential to affect the outcome of coevolution.

Characterization of Pinus spp. needles by gas chromatography and mass spectrometry: Application to plant-insect interactions

Dissertação apresentada para obtenção do Grau de Doutor em Ciências do Ambiente pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecn

Fifty years after Ehrlich and Raven, is there support for plant-insect coevolution as a major driver of species diversification?

Since Ehrlich & Raven's seminal paper 50 years ago, coevolution has been seen as a major driver of species diversification. Here, we review classical and more recent case studies on the coevolution of plants and associated insects, to examine whether the coevolutionary component holds as an explanation of their current diversity. We discuss the main dogmas in coevolution and argue that coevolutionary processes should not be considered as major drivers of diversification in plants and insects. Instead, we suggest that coevolution essentially occurs through relatively short 'interludes', making the pattern difficult to detect. We also criticize the use of comparative phylogenetics to investigate coevolutionary processes, as coevolution may not necessarily produce congruent phylogenies among interacting lineages and, in turn, other processes may produce patterns of codivergence. Finally, we propose new lines of investigation for future research.

An extreme case of plant-insect co-diversification: figs and fig-pollinating wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale co-phylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on average, wasps had sequences from 77% of six genes (5.6kb), figs had sequences from 60% of five genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based co-phylogenetic analyses further support the co-diversification hypothesis. Biogeographic analyses indicate that the presentday distribution of fig and pollinator lineages is consistent with an Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term co-diversification.

Serpentine leafminer (Liriomyza trifolii) on potato (Solanum tuberosum): field observations and plant photosynthetic responses to injury

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Size and lipid content of nonmyrmecochorous diaspores: Effects on the interaction with litter-foraging ants in the Atlantic rain forest of Brazil

Ants are often attracted to diaspores not adapted for dispersal by ants. These diaspores may occasionally benefit from this interaction. We selected six nonmyrmecochorous plant species (Virola oleifera, Eugenia stictosepala, Cabralea canjerana, Citharexylum myrianthum, Alchornea glandulosa and Hyeronima alchorneoides) whose diaspores differ in size and lipid content, and investigated how these features affect the outcome of ant-diaspore interactions on the floor of a lowland Atlantic forest of Southeast Brazil. A total of 23 ant species were seen interacting with diaspores on the forest floor. Ants were generally rapid at discovering and cleaning the diaspore pulp or aril. Recruitment rate and ant attendance were higher for lipid-rich diaspores than for lipid-poor ones. Removal rate and displacement distance were higher for small diaspores. The large ponerine ant Pachycondyla striata, one of the most frequent attendants to lipid-rich arillate diaspores, transported the latter into their nests and discarded clean intact seeds on refuse piles outside the nest. Germination tests with cleaned and uncleaned diaspores revealed that the removal of pulp or aril may increase germination success in Virola oleifera, Cabralea canjerana, Citharexylum myrianthum and Alchornea glandulosa. Gas chromatography analyses revealed a close similarity in the fatty acid composition of the arils of the lipid-rich diaspores and the elaiosome of a typical myrmecochorous seed (Ricinus communis), corroborating the suggestion that some arils and elaiosomes are chemically similar. Although ant-derived benefits to diaspores - secondary dispersal and/or increased germination - varied among the six plant species studied, the results enhanced the role of ant-diaspore interactions in the post-dispersal fates of nonmyrmecochorous seeds in tropical forests. The size and the lipid-content of the diaspores were shown to be major determinants of the outcome of such interactions.

The role of multiple partners in a digestive mutualism with a protocarnivorous plant

Background and aimsThe protocarnivorous plant Paepalanthus bromelioides (Eriocaulaceae) is similar to bromeliads in that this plant has a rosette-like structure that allows rainwater to accumulate in leaf axils (i.e. phytotelmata). Although the rosettes of P. bromelioides are commonly inhabited by predators (e.g. spiders), their roots are wrapped by a cylindrical termite mound that grows beneath the rosette. In this study it is predicted that these plants can derive nutrients from recycling processes carried out by termites and from predation events that take place inside the rosette. It is also predicted that bacteria living in phytotelmata can accelerate nutrient cycling derived from predators.MethodsThe predictions were tested by surveying plants and animals, and also by performing field experiments in rocky fields from Serra do Cipó, Brazil, using natural abundance and enriched isotopes of 15N. Laboratory bioassays were also conducted to test proteolytic activities of bacteria from P. bromelioides rosettes.Key ResultsAnalyses of 15N in natural nitrogen abundances showed that the isotopic signature of P. bromelioides is similar to that of carnivorous plants and higher than that of non-carnivorous plants in the study area. Linear mixing models showed that predatory activities on the rosettes (i.e. spider faeces and prey carcass) resulted in overall nitrogen contributions of 26·5 % (a top-down flux). Although nitrogen flux was not detected from termites to plants via decomposition of labelled cardboard, the data on 15N in natural nitrogen abundance indicated that 67 % of nitrogen from P. bromelioides is derived from termites (a bottom-up flux). Bacteria did not affect nutrient cycling or nitrogen uptake from prey carcasses and spider faeces.ConclusionsThe results suggest that P. bromelioides derive nitrogen from associated predators and termites, despite differences in nitrogen cycling velocities, which seem to have been higher in nitrogen derived from predators (leaves) than from termites (roots). This is the first study that demonstrates partitioning effects from multiple partners in a digestion-based mutualism. Despite most of the nitrogen being absorbed through their roots (via termites), P. bromelioides has all the attributes necessary to be considered as a carnivorous plant in the context of digestive mutualism. © 2012 The Author. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.

Natural history of an ant-plant-butterfly interaction in a Neotropical savanna

Non-myrmecophilous lepidopteran larvae using plants bearing ant attractants such as extrafloral nectaries are good models for studying morphological and behavioural mechanisms against ant predation. Udranomia spitzi (Hesperiidae) is a butterfly whose larvae feed on leaves of Ouratea spectabilis (Ochnaceae), a plant with extrafloral nectaries. We described the early stages of U. spitzi, and used field observations and experiments to investigate the defensive strategies of caterpillars against predatory ants. Larvae pass through five instars and pupation occurs inside larval leaf shelters. Ant-exclusion experiments revealed that the presence of ants did not affect significantly caterpillar survival. Predation experiments showed that vulnerability to ant predation decreased with increase in larval size. The present study showed that predatory ants are not as relevant as demonstrated for other systems, and also illustrates how observational data and field experiments can contribute to a better understanding of the biology and ecology of a species of interest.

An Extreme Case of Plant-Insect Codiversification: Figs and Fig-Pollinating Wasps

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification.

Where Is My Food? Brazilian Flower Fly Steals Prey from Carnivorous Sundews in a Newly Discovered Plant-Animal Interaction

A new interaction between insects and carnivorous plants is reported from Brazil. Larvae of the predatory flower fly Toxomerus basalis (Diptera: Syrphidae: Syrphinae) have been found scavenging on the sticky leaves of several carnivorous sundew species (Drosera, Droseraceae) in Minas Gerais and São Paulo states, SE Brazil. This syrphid apparently spends its whole larval stage feeding on prey trapped by Drosera leaves. The nature of this plant-animal relationship is discussed, as well as the Drosera species involved, and locations where T. basalis was observed. 180 years after the discovery of this flower fly species, its biology now has been revealed. This is (1) the first record of kleptoparasitism in the Syrphidae, (2) a new larval feeding mode for this family, and (3) the first report of a dipteran that shows a kleptoparasitic relationship with a carnivorous plant with adhesive flypaper traps. The first descriptions of the third instar larva and puparium of T. basalis based on Scanning Electron Microscope analysis are provided.

Temperature and the synchrony of plant-insect interactions

Increasing temperatures resulting from climate change have within recent years been shown to advance phenological events in a large number of species worldwide. Species can differ in their response to increasing temperatures, and understanding the mechanisms that determine the response is therefore of great importance in order to understand and predict how a warming climate can influence both individual species, but also their interactions with each other and the environment. Understanding the mechanisms behind responses to increasing temperatures are however largely unexplored. The selected study system consisting of host plant species of the Brassicaceae family and their herbivore Anthocharis cardamines, is assumed to be especially vulnerable to climatic variations. Through the use of this study system, the aim of this thesis is to study differences in the effect of temperature on development to start of flowering within host plant species from different latitudinal regions (study I), and among host plant species (study II). We also investigate whether different developmental phases leading up to flowering differ in sensitivity to temperature (study II), and if small-scale climatic variation in spring temperature influence flowering phenology and interactions with A. cardamines (study III). Finally, we investigate if differences in the timing of A. cardamines relative to its host plants influence host species use and the selection of host individuals differing in phenology within populations (study IV). Our results showed that thermal reaction norms differ among regions along a latitudinal gradient, with the host plant species showing a mixture of co-, counter- and mixed gradient patterns (study I). We also showed that observed differences in the host plant species order of flowering among regions and years might be caused by both differences in the distribution of warm days during development and differences in the sensitivity to temperature in different phases of development (study II). In addition, we showed that small-scale variations in temperature led to variation in flowering phenology among and within populations of C. pratensis, impacting the interactions with the butterfly herbivore A. cardamines. Another result was that the less the mean plant development stage of a given plant species in the field deviated from the stage preferred by the butterfly for oviposition, the more used was the species as a host by the butterfly (study IV). Finally, we showed that the later seasonal appearance of the butterflies relative to their host plants, the higher butterfly preference for host plant individuals with a later phenology, corresponding to a preference for host plants in earlier development stages (study IV). For our study system, this thesis suggest that climate change will lead to changes in the interactions between host plants and herbivore, but that differences in phenology among host plants combined with changes in host species use of the herbivore might buffer the herbivore against negative effects of climate change. Our work highlights the need to understand the mechanisms behind differences in the responses of developmental rates to temperature between interacting species, as well as the need to account for differences in temperature response for interacting organisms from different latitudinal origins and during different developmental phases in order to understand and predict the consequences of climate change. 

Study of Rapid Alkalinization Factor (RALF)genes as plant susceptibility agents during plant-pathogen interaction in strawberry

Rapid Alkalinization Factor (RALF) are cysteins-rich peptides ubiquitous in plant kingdom. They play multiple roles as hormone signals and recently their involvement in host-pathogen crosstalk as negative regulator of immunity in Arabidopsis has also been recognized. In addition, RALF homologue peptides are secreted by different fungal pathogens as effectors during early stages of infections. The aim of this work was to characterize RALF genes as susceptibility factors during plant pathogen interaction in strawberry. For this, the genomic organization of the RALF gene families in the octoploid strawberry (Fragaria × ananassa) and the re-annotated genome of Fragaria vesca were described , identifying 13 member in F. vesca (FvRALF) and 50 members in F. x ananassa (FaRALF). The changes in expression of fruit FaRALF genes was investigated upon infection with C.acutatum and B. cinerea showing that, among RALF genes expressed in fruit, FaRALF3 was the only one upregulated by fungal infection in the ripe stage. A role of FaRALF3 as susceptibility gene was then assessed trough Agrobacterium-mediated transient FaRALF3 overexpression and silencing in fruits, revealing that FaRALF3 expression promotes fungal growth and hyphae penetration in host tissues. In silico analysis was used to identify distinct pathogen inducible elements upstream of the FaRALF3 gene. Agroinfiltration of strawberry fruit with deletion constructs of the FaRALF3 promoter identified a 5’ region required for FaRALF3 expression in fruit, but failed to identify a region responsible for fungal induced expression. Furthermore, FaRALF3 and strawberry receptor FERONIA (FaMRLK47) were heterologously expressed in E. coli in order to purify active proteins forms and study RALF-FERONIA interaction in strawberry. However, it was not possible to obtain pure and active proteins. Finally RNAi transgenic plants silenced for the FvRALF13 gene were genotypically and phenotypically characterized suggesting a role of FvRALF13 in flowering time regulation and reproductive organs development.

Molecular characterization of a miraculin-like gene differentially expressed during coffee development and coffee leaf miner infestation

The characterization of a coffee gene encoding a protein similar to miraculin-like proteins, which are members of the plant Kunitz serine trypsin inhibitor (STI) family of proteinase inhibitors (PIs), is described. PIs are important proteins in plant defence against insects and in the regulation of proteolysis during plant development. This gene has high identity with the Richadella dulcifica taste-modifying protein miraculin and with the tomato protein LeMir; and was named as CoMir (Coffea miraculin). Structural protein modelling indicated that CoMir had structural similarities with the Kunitz STI proteins, but suggested specific folding structures. CoMir was up-regulated after coffee leaf miner (Leucoptera coffella) oviposition in resistant plants of a progeny derived from crosses between C. racemosa (resistant) and C. arabica (susceptible). Interestingly, this gene was down-regulated during coffee leaf miner herbivory in susceptible plants. CoMir expression was up-regulated after abscisic acid application and wounding stress and was prominent during the early stages of flower and fruit development. In situ hybridization revealed that CoMir transcripts accumulated in the anther tissues that display programmed cell death (tapetum, endothecium and stomium) and in the metaxylem vessels of the petals, stigma and leaves. In addition, the recombinant protein CoMir shows inhibitory activity against trypsin. According to the present results CoMir may act in proteolytic regulation during coffee development and in the defence against L. coffeella. The similarity of CoMir with other Kunitz STI proteins and the role of CoMir in plant development and plant stress are discussed.