Consequences of plant population size for pollinator visitation and plant reproductive success

Habitat loss and fragmentation have a prominent role in determining the size of plant populations, and can affect plant-pollinator interactions. It is hypothesized that in small plant populations the ability to set seeds can be reduced due to limited pollination services, since individuals in small populations can receive less quantity or quality of visits. In this study, I investigated the effect of population size on plant reproductive success and insect visitation in 8 populations of two common species in the island of Lesvos, Greece (Mediterranean Sea), Echium plantagineum and Ballota acetabulosa, and of a rare perennial shrub endemic to north-central Italy, Ononis masquillierii. All the three species depended on insect pollinators for sexual reproduction. For each species, pollen limitation was present in all or nearly all populations, but the relationship between pollen limitation and population size was only present in Ononis masquillierii. However, in Echium plantagineum, significant relationships between both open-pollinated and handcrossed-pollinated seed sets and population size were found, being small populations comparatively less productive than large ones. Additionally, for this species, livestock grazing intensity was greater for small populations and for sparse patches, and had a negative influence on productivity of the remnant plants. Both Echium plantagineum and Ballota acetabulosa attracted a great number of insects, representing a wide spectrum of pollinators, thereby can be considered as generalist species. For Ballota acetabulosa, the most important pollinators were megachilid female bees, and insect diversity didn’t decrease with decreasing plant population size. By contrast, Ononis masquillierii plants generally received few visits, with flowers specialized on small bees (Lasioglossum spp.), representing the most important insect guild. In Echium plantagineum and Ballota acetabulosa, plants in small and large populations received the same amount of visits per flower, and no differences in the number of intraplant visited flowers were detected. On the contrary, large Ononis populations supported higher amounts of pollinators than small ones. At patch level, high Echium flower density was associated with more and higher quality pollinators. My results indicate that small populations were not subject to reduced pollination services than large ones in Echium plantagineum and Ballota acetabulosa, and suggest that grazing and resource limitation could have a major impact on population fitness in Echium plantagineum. The absence of any size effects in these two species can be explained in the light of their high local abundance, wide habitat specificity, and ability to compete with other co-flowering species for pollinators. By contrast, size represents a key characteristic for both pollination and reproduction in Ononis masquillierii populations, as an increase in size could mitigate the negative effects coming from the disadvantageous reproductive traits of the species. Finally, the widespread occurrence of pollen limitation in the three species may be the result of 1) an ongoing weakening or disruption of plantpollinator interactions derived from ecological perturbations, 2) an adaptive equilibrium in response to stochastic processes, and 3) the presence of unfavourable reproductive traits (for Ononis masquillierii).

B-type cytochromes of the DOMON domain superfamily (Novel redox elements of plant plasma membrane)

The aim of the present study is understanding the properties of a new group of redox proteins having in common a DOMON-type domain with characteristics of cytochromes b. The superfamily of proteins containing a DOMON of this type includes a few protein families. With the aim of better characterizing this new protein family, the present work addresses both a CyDOM protein (a cytochrome b561) and a protein only comprised of DOMON(AIR12), both of plant origin. Apoplastic ascorbate can be regenerated from monodehydroascorbate by a trans-plasma membrane redox system which uses cytosolic ascorbate as a reductant and comprises a high potential cytochrome b. We identified the major plasma membrane (PM) ascorbate-reducible b-type cytochrome of bean (Phaseolus vulgaris) and soybean (Glycine max) hypocotyls as orthologs of Arabidopsis auxin-responsive gene air12. The protein, which is glycosylated and glycosylphosphatidylinositol-anchored to the external side of the PM in vivo, was expressed in Pichia pastoris in a recombinant form, lacking the glycosylphosphatidylinositol-modification signal, and purified from the culture medium. Recombinant AIR12 is a soluble protein predicted to fold into a β-sandwich domain and belonging to the DOMON superfamily. It is shown to be a b-type cytochrome with a symmetrical α-band at 561 nm, to be fully reduced by ascorbate and fully oxidized by monodehydroascorbate. Redox potentiometry suggests that AIR12 binds two high-potential hemes (Em,7 +135 and +236 mV). Phylogenetic analyses reveal that the auxin-responsive genes AIR12 constitute a new family of plasma membrane b-type cytochromes specific to flowering plants. Although AIR12 is one of the few redox proteins of the PM characterized to date, the role of AIR12 in trans-PM electron transfer would imply interaction with other partners which are still to be identified. Another part of the present project was aimed at understanding of a soybean protein comprised of a DOMON fused with a well-defined b561 cytochrome domain (CyDOM). Various bioinformatic approaches show this protein to be composed of an N-terminal DOMON followed by b561 domain. The latter contains five transmembrane helices featuring highly conserved histidines, which might bind haem groups. The CyDOM has been cloned and expressed in the yeast Pichia pastoris, and spectroscopic analyses have been accomplished on solubilized yeast membranes. CyDOM clearly reveal the properties of b-type cytochrome. The results highlight the fact that CyDOM is clearly able to lead an electron flux through the plasmamembrane. Voltage clamp experiments demonstrate that Xenopus laevis oocytes transformed with CyDOM of soybean exhibit negative electrical currents in presence of an external electron acceptor. Analogous investigations were carried out with SDR2, a CyDOM of Drosophila melanogaster which shows an electron transport capacity even higher than plant CyDOM. As quoted above, these data reinforce those obtained in plant CyDOM on the one hand, and on the other hand allow to attribute to SDR2-like proteins the properties assigned to CyDOM. Was expressed in Regenerated tobacco roots, transiently transformed with infected a with chimeral construct GFP: CyDOM (by A. rhizogenes infection) reveals a plasmamembrane localization of CyDOM both in epidermal cells of the elongation zone of roots and in root hairs. In conclusion. Although the data presented here await to be expanded and in part clarified, it is safe to say they open a new perspective about the role of this group of proteins. The biological relevance of the functional and physiological implications of DOMON redox domains seems noteworthy, and it can but increase with future advances in research. Beyond the very finding, however interesting in itself, of DOMON domains as extracellular cytochromes, the present study testifies to the fact that cytochrome proteins containing DOMON domains of the type of “CyDOM” can transfer electrons through membranes and may represent the most important redox component of the plasmamembrane as yet discovered.

The Domon Family of Plant Plasma Membrane B-Type Cytochromes: Heterologous Expression, Biochemical Characterization and Physiological Roles in Vivo

The DOMON domain is a domain widespread in nature, predicted to fold in a β-sandwich structure. In plants, AIR12 is constituted by a single DOMON domain located in the apoplastic space and is GPI-modified for anchoring to the plasma membrane. Arabidopsis thaliana AIR12 has been heterologously expressed as a recombinant protein (recAtAIR12) in Pichia pastoris. Spectrophotometrical analysis of the purified protein showed that recAtAir12 is a cytochrome b. RecAtAIR12 is highly glycosylated, it is reduced by ascorbate, superoxide and naftoquinones, oxidised by monodehydroascorbate and oxygen and insensitive to hydrogen peroxide. The addition of recAtAIR12 to permeabilized plasma membranes containing NADH, FeEDTA and menadione, caused a statistically significant increase in hydroxyl radicals as detected by electron paramagnetic resonance. In these conditions, recAtAIR12 has thus a pro-oxidant role. Interestingly, AIR12 is related to the cytochrome domain of cellobiose dehydrogenase which is involved in lignin degradation, possibly via reactive oxygen species (ROS) production. In Arabidopsis the Air12 promoter is specifically activated at sites where cell separations occur and ROS, including •OH, are involved in cell wall modifications. air12 knock-out plants infected with Botrytis cinerea are more resistant than wild-type and air12 complemented plants. Also during B. cinerea infection, cell wall modifications and ROS are involved. Our results thus suggest that AIR12 could be involved in cell wall modifying reactions by interacting with ROS and ascorbate. CyDOMs are plasma membrane redox proteins of plants that are predicted to contain an apoplastic DOMON fused with a transmembrane cytochrome b561 domain. CyDOMs have never been purified nor characterised. The trans-membrane portion of a soybean CyDOM was expressed in E. coli but purification could not be achieved. The DOMON domain was expressed in P. pastoris and shown to be itself a cytochrome b that could be reduced by ascorbate.

Use of Biotechnology to increase the content of bioactive compounds in fermented foods of plant origin

The objectives of this PhD research were: i) to evaluate the use of bread making process to increase the content of β-glucans, resistant starch, fructans, dietary fibers and phenolic compounds of kamut khorasan and wheat breads made with flours obtained from kernels at different maturation stage (at milky stage and fully ripe) and ii) to study the impact of whole grains consumption in the human gut. The fermentation and the stages of kernel development or maturation had a great impact on the amount of resistant starch, fructans and β-glucans as well as their interactions resulted highly statistically significant. The amount of fructans was high in kamut bread (2.1g/100g) at the fully ripe stage compared to wheat during industrial fermentation (baker’s yeast). The sourdough increases the content of polyphenols more than industrial fermentation especially in bread made by flour at milky stage. From the analysis of volatile compounds it resulted that the sensors of electronic nose perceived more aromatic compound in kamut products, as well as the SPME-GC-MS, thus we can assume that kamut is more aromatic than wheat, so using it in sourdough process can be a successful approach to improve the bread taste and flavor. The determination of whole grain biormakers such as alkylresorcinols and others using FIE-MS AND GC-tof-MS is a valuable alternative for further metabolic investigations. The decrease of N-acetyl-glucosamine and 3-methyl-hexanedioic acid in kamut faecal samples suggests that kamut can have a role in modulating mucus production/degradation or even gut inflammation. This work gives a new approach to the innovation strategies in bakery functional foods, that can help to choose the right or best combination between stages of kernel maturation-fermentation process and baking temperature.

Influence of plant structure, cultural pratices and environmental conditions on the development of the bacterial canker of kiwifruits

Italy has a preeminent rank in kiwifruit industry, being the first exporter and the second largest producer after China. However, in the last years kiwifruit yields and the total cultivated area considerably decreased, due to the pandemic spread of the bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). Several climatic conditions and cultural practices affect the development of the bacterial canker. This research work focused on the impact of agricultural practices and microclimate conditions on the incidence and epidemiology of Psa in the orchard. Therefore, the effect of fertilization, irrigation, use of bio-regulators, rootstock, training system and pruning were examined. The effect of different tunnel systems was analyzed as well, to study the plant-pathogen interaction. Considering the importance of insects as vectors in other pathosystems, the role of Metcalfa pruinosa in the spread of the bacterial canker was investigated in controlled conditions. In addition, quality and storage properties of fruits from infected plants were assessed. The study of all these aspects of the agronomic practices is useful to define a strategy to limit the bacterial diffusion in the orchard. Overall, excess nitrogen fertilization, water stress, stagnant water supplies, pruning before summer and the high number of Metcalfa pruinosa increased the Psa incidence. In contrast, tunnel covers may be useful for the control of the disease, with special attention to the kind of material.

Organic fertilization of peach trees: implication on nitrogen availability, root growth and carbon distribution within plant

In the last years, sustainable horticulture has been increasing; however, to be successful this practice needs an efficient soil fertility management to maintain a high productivity and fruit quality standards. For this purpose composted organic materials from agri-food industry and municipal solid waste has been used as a source to replace chemical fertilizers and increase soil organic matter. To better understand the influence of compost application on soil fertility and plant growth, we carried out a study comparing organic and mineral nitrogen (N) fertilization in micro propagated plants, potted trees and commercial peach orchard with these aims: 1. evaluation of tree development, CO2 fixation and carbon partition to the different organs of two-years-old potted peach trees. 2. Determination of soil N concentration and nitrate-N effect on plant growth and root oxidative stress of micro propagated plant after increasing rates of N applications. 3. Assessment of soil chemical and biological fertility, tree growth and yield and fruit quality in a commercial orchard. The addition of compost at high rate was effective in increasing CO2 fixation, promoting root growth, shoot and fruit biomass. Furthermore, organic fertilizers influenced C partitioning, favoring C accumulation in roots, wood and fruits. The higher CO2 fixation was the result of a larger tree leaf area, rather than an increase in leaf photosynthetic efficiency, showing a stimulation of plant growth by application of compost. High concentrations of compost increased total soil N concentration, but were not effective in increasing nitrate-N soil concentration; in contrast mineral-N applications increased linearly soil nitrate-N, even at the lowest rate tested. Soil nitrate-N concentration influenced positively plant growth at low rate (60- 80 mg kg-1), whereas at high concentrations showed negative effects. In this trial, the decrease of root growth, as a response to excessive nitrate-N soil concentration, was not anticipated by root oxidative stress. Continuous annual applications of compost for 10 years enhanced soil organic matter content and total soil N concentration. Additionally, high rate of compost application (10 t ha-1 year-1) enhanced microbial biomass. On the other hand, different fertilizers management did not modify tree yield, but influenced fruit size and precocity index. The present data support the idea that organic fertilizers can be used successfully as a substitute of mineral fertilizers in fruit tree nutrient management, since they promote an increase of soil chemical and biological fertility, prevent excessive nitrate-N soil concentration, promote plant growth and potentially C sequestration into the soil.

Pathogenesis of cell toxicity by plant toxins

Ribosome inactivating proteins (RIPs) are a family of plant proteins that depurinate the major rRNA, inhibiting the protein synthesis. RIPs are divided into type 1, single chain proteins with enzymatic activity, and type 2 RIPs (toxic and non-toxic), with the enzymatic chain linked to a binding chain. RIPs have been used alone or as toxic component of immunotoxins for experimental therapy of many diseases. The knowledge of cell death pathway(s) induced by RIPs could be useful for clarifying the mechanisms induced by RIPs and for designing specific immunotherapy. The topic of the current study was (i) the determination of the amino acid sequence of the type 2 RIP stenodactylin. The comparison with other RIPs showed that the A chain is related to other toxic type 2 RIPs. whereas the B chain is more related to the non-toxic type 2 RIPs. This latter result is surprising because stenodactylin is actually the most toxic type 2 RIP known; (ii) the study of the cell death mechanisms induced by stenodactylin in human neuroblastoma cells (NB100). High doses of stenodactylin can activate the effector caspases (perhaps through the DNA damage and/or intrinsic/extrinsic pathways) and also cause ROS generation. Low doses cause a caspase-dependent apoptosis, mainly via extrinsic pathway. Moreover, the activation of caspases precedes the inhibition of protein synthesis; (iii) the investigation of the cell death pathway induced by the non-toxic type 2 RIPs ebulin l and nigrin b. These RIPs demonstrated high enzymatic activity in a cell-free system, but they lack high cytotoxicity. These preliminary studies demonstrate that the cell death mechanism induced by the two non-toxic RIPs is partially caspase-dependent apoptosis, but other mechanisms seem to be involved

Soil application of Meliaceae derivatives: effect on carbon and nitrogen dynamics in the soil-plant system

The effect of soil incorporation of 7 Meliaceae derivatives (6 commercial neem cakes and leaves of Melia azedarach L.) on C and N dynamics and on nutrient availability to micropropagated GF677 rootstock was investigated. In a first laboratory incubation experiment the derivatives showed different N mineralization dynamics, generally well predicted by their C:N ratio and only partly by their initial N concentration. All derivatives increased microbial biomass C, thus representing a source of C for the soil microbial population. Soil addition of all neem cakes (8 g kg-1) and melia leaves (16 g kg-1) had a positive effect on plant growth and increased root N uptake and leaf green colour of micropropagated plants of GF677. In addition, the neem cakes characterized by higher nutrient concentration increased P and K concentration in shoot and leaves 68 days after the amendment. In another experiment, soil incorporation of 15N labeled melia leaves (16 g kg-1) had no effect on the total amount of plant N, however the percentage of melia derived-N of treated plants ranged between 0.8% and 34% during the experiment. At the end of the growing season, about 7% of N added as melia leaves was recovered in plant, while 70% of it was still present in soil. Real C mineralization and the priming effect induced by the addition of the derivatives were quantified by a natural 13C abundance method. The real C mineralization of the derivatives ranged between 22% and 40% of added-C. All the derivatives studied induced a positive priming effect and, 144 days after the amendment, the amount of C primed corresponded to 26% of added-C, for all the derivatives. Despite this substantial priming effect, the C balance of the soil, 144 days after the amendment, always resulted positive.

Relationships between plant diversity and environmental heterogeneity in rupicolous grasslands on gypsum. The case study of Alysso-Sedion albi (Habitat 6110).

Plant communities on weathered rock and outcrops are characterized by high values in species richness (Dengler 2006) and often persist on small and fragmented surfaces. Yet very few studies have examined the relationships between heterogeneity and plant diversity at small scales, in particular in poor-nutrient and low productive environment (Shmida and Wilson 1985, Lundholm 2003). In order to assess these relationships both in space and time in relationship, two different approaches were employed in the present study, in two gypsum outcrops of Northern Apennine. Diachronic and synchronic samplings from April 2012 to March 2013 were performed. A 50x50 cm plot was used in both samplings such as the sampling unit base. The diachronic survey aims to investigate seasonal patterning of plant diversity by the use of images analysis techniques integrated with field data and considering also seasonal climatic trend, the substrate quality and its variation in time. The purpose of the further, synchronic sampling was to describe plant diversity pattern as a function of the environmental heterogeneity meaning in substrate typologies, soil depth and topographic features. Results showed that responses of diversity pattern depend both on the resources availability, environmental heterogeneity and the manner in which the different taxonomic group access to them during the year. Species richness and Shannon diversity were positively affected by increasing in substrate heterogeneity. Furthermore a good turnover in seasonal species occurrence was detected. This vegetation may be described by the coexistence of three groups of species which created a gradient from early colonization stages, characterized by greater slope and predominance of bare rock, gradually to situation of more developed soil.