Development of pollinated and unpollinated ovules in Ginkgo biloba: unravelling the role of pollen in ovule tissue maturation.

In gymnosperms such as Ginkgo biloba, the arrival of pollen plays a key role in ovule development, before fertilization occurs. Accordingly, G. biloba female plants geographically isolated from male plants abort all their ovules after the pollination drop emission, which is the event that allows the ovule to capture pollen grains. To decipher the mechanism induced by pollination required to avoid ovule senescence and then abortion, we compared the transcriptomes of pollinated and unpollinated ovules at three time points after the end of the emission of pollination drop. Transcriptomic and in situ expression analyses revealed that several key genes involved in programmed cell death such as senescence and apoptosis, DNA replication, and cell cycle regulation were differentially expressed in unpollinated ovules compared to pollinated ovules. We provide evidence that the pollen captured by the pollination drop affects auxin local accumulation and might cause deregulation of key genes required for the ovule's programmed cell death, activating both the cell cycle regulation and DNA replication genes.

Response of Coccomyxa cimbrica sp.nov. to Increasing Doses of Cu(II) as a Function of Time: Comparison between Exposure in a Microfluidic Device or with Standard Protocols.

In this study, we explore how the in vitro conditions chosen to cultivate and observe the long-term (up to 72 h) toxic effect of Cu(II) on the freshwater microalga Coccomyxa cimbrica sp.nov. can affect the dose response in time. We test three different cultivation protocols: (i) under static conditions in sealed glass cells, (ii) in a microfluidic device, where the sample is constantly circulated with a peristaltic pump, and (iii) under continuous agitation in plastic falcons on an orbital shaker. The advantage and novelty of this study resides in the fact that each condition can mimic different environmental conditions that alga cells can find in nature. The effect of increasing dose of Cu(II) as a function of time (24, 48, and 72 h) is monitored following chlorophyll a fluorescence intensity from single cells. Fluorescence lifetime imaging experiments are also explored to gain information on the changes induced by Cu(II) in the photosynthetic cycle of this microalga.

Identification of key regulatory genes involved in the sporophyte and gametophyte development in Ginkgo biloba ovules revealed by in situ expression analyses.

PREMISE: In Arabidopsis thaliana, the role of the most important key genes that regulate ovule development is widely known. In nonmodel species, and especially in gymnosperms, the ovule developmental processes are still quite obscure. In this study, we describe the putative roles of Ginkgo biloba orthologs of regulatory genes during ovule development. Specifically, we studied AGAMOUS (AG), AGAMOUS-like 6 (AGL6), AINTEGUMENTA (ANT), BELL1 (BEL1), Class III HD-Zip, and YABBY Ginkgo genes. METHODS: We analyzed their expression domains through in situ hybridizations on two stages of ovule development: the very early stage that corresponds to the ovule primordium, still within wintering buds, and the late stage at pollination time. RESULTS: GBM5 (Ginkgo ortholog of AG), GbMADS8 (ortholog of AGL6) and GbC3HDZ1-2-3 were expressed in both the stages of ovule development, while GbMADS1, GbAGL6-like genes (orthologs of AGL6), GbBEL1-2 and YABBY Ginkgo orthologs (GbiYAB1B and GbiYABC) seem mostly involved at pollination time. GbANTL1 was not expressed in the studied stages and was different from GbANTL2 and GbBEL1, which seem to be involved at both stages of ovule development. In Ginkgo, the investigated genes display patterns of expression only partially comparable to those of other studied seed plants. CONCLUSIONS: The expression of most of these regulatory genes in the female gametophyte region at pollination time leads to suggest a communication between the sporophytic maternal tissue and the developing female gametophyte, as demonstrated for well-studied model angiosperms.

Characterization of bacterial communities isolated from municipal waste compost and screening of their plant-interactive phenotypes.

Four batches of commercial compost obtained from the organic fraction of municipal solid waste were analyzed from chemical and microbiological standpoints. The working hypothesis was that, being this type of compost derived partly from plant waste, it could contain plant-growth promoting bacterial endophytes, prone to be active again upon its usual delivery as fertilizer. Culturable bacteria were isolated at different temperatures, quantified by colony morphology, identified taxonomically by 16S sequencing and screened for plant-growth promoting phenotypes including auxin and siderophore production, phosphate solubilization and peptide mineralization to ammonia. In parallel, the total community was assessed by culture independent DNA metabarcoding. The capability of plants to select, uptake and internally multiply bacteria from these compost samples was analyzed using grapevine in-vitro rooting cuttings from which acquired bacteria were reisolated, quantified and their identities determined as above. Major differences in compost bacterial composition were observed as function of the season, with the winter sample being rather distinct from the summer ones. Bacillales and Actinomycetales dominated the culturable communities while Alteromonadales, Oceanospirillales and Flavobacteriales prevailed in the total community. In spite of the challenging composting cycle conditions, the plant nature of the main input substrates appeared determinant in guaranteeing that 82% of the culturable bacteria were found endowed with one or more of the plant growth-promoting phenotypes tested. Beside its fertilization role, compost proved to be also a potential inoculant carrier for the in-soil delivery of plant beneficial microorganisms. Furthermore, upon an in vitro passage through grapevine plants under axenic conditions, the subsequently recoverable endophyte community yielded also members of the Rhizobiales order which had not been detectable when culturing directly from compost. This observation further suggests that compost-borne plant-interacting taxa could be also rescued from non-culturable states and/or enriched above detectability levels by a contact with their potential host plants.

Monitoring calcium handling by the plant endoplasmic reticulum with a low-Ca2+ -affinity targeted aequorin reporter.

Precise measurements of dynamic changes in free Ca2+ concentration in the lumen of the plant endoplasmic reticulum (ER) have been lacking so far, despite increasing evidence for the contribution of this intracellular compartment to Ca2+ homeostasis and signalling in the plant cell. In the present study, we targeted an aequorin chimera with reduced Ca2+ affinity to the ER membrane and facing the ER lumen. To this aim, the cDNA for a low-Ca2+ -affinity aequorin variant (AEQmut) was fused to the nucleotide sequence encoding a non-cleavable N-terminal ER signal peptide (fl2). The correct targeting of fl2-AEQmut was confirmed by immunocytochemical analyses in transgenic Arabidopsis thaliana (Arabidopsis) seedlings. An experimental protocol well-established in animal cells - consisting of ER Ca2+ depletion during photoprotein reconstitution followed by ER Ca2+ refilling - was applied to carry out ER Ca2+ measurements in planta. Rapid and transient increases of the ER luminal Ca2+ concentration ([Ca2+ ]ER ) were recorded in response to different environmental stresses, displaying stimulus-specific Ca2+ signatures. The comparative analysis of ER and chloroplast Ca2+ dynamics indicates a complex interplay of these organelles in shaping cytosolic Ca2+ signals during signal transduction events. Our data highlight significant differences in basal [Ca2+ ]ER and Ca2+ handling by plant ER compared to the animal counterpart. The set-up of an ER-targeted aequorin chimera extends and complements the currently available toolkit of organelle-targeted Ca2+ indicators by adding a reporter that improves our quantitative understanding of Ca2+ homeostasis in the plant endomembrane system.

Expression and Functional Analyses of Nymphaea caerulea MADS-Box Genes Contribute to Clarify the Complex Flower Patterning of Water Lilies.

Nymphaeaceae are early diverging angiosperms with large flowers characterized by showy petals and stamens not clearly whorled but presenting a gradual morphological transition from the outer elements to the inner stamens. Such flower structure makes these plant species relevant for studying flower evolution. MADS-domain transcription factors are crucial components of the molecular network that controls flower development. We therefore isolated and characterized MADS-box genes from the water lily Nymphaea caerulea. RNA-seq experiments on floral buds have been performed to obtain the transcript sequences of floral organ identity MADS-box genes. Maximum Likelihood phylogenetic analyses confirmed their belonging to specific MADS-box gene subfamilies. Their expression was quantified by RT-qPCR in all floral organs at two stages of development. Protein interactions among these transcription factors were investigated by yeast-two-hybrid assays. We found especially interesting the involvement of two different AGAMOUS-like genes (NycAG1 and NycAG2) in the water lily floral components. They were therefore functionally characterized by complementing Arabidopsis ag and shp1 shp2 mutants. The expression analysis of MADS-box genes across flower development in N. caerulea described a complex scenario made of numerous genes in numerous floral components. Their expression profiles in some cases were in line with what was expected from the ABC model of flower development and its extensions, while in other cases presented new and interesting gene expression patterns, as for instance the involvement of NycAGL6 and NycFL. Although sharing a high level of sequence similarity, the two AGAMOUS-like genes NycAG1 and NycAG2 could have undergone subfunctionalization or neofunctionalization, as only one of them could partially restore the euAG function in Arabidopsis ag-3 mutants. The hereby illustrated N. caerulea MADS-box gene expression pattern might mirror the morphological transition from the outer to the inner floral organs, and the presence of transition organs such as the petaloid stamens. This study is intended to broaden knowledge on the role and evolution of floral organ identity genes and the genetic mechanisms causing biodiversity in angiosperm flowers.

The role of pollination in controlling Ginkgo biloba ovule development.

Generally, in gymnosperms, pollination and fertilization events are temporally separated and the developmental processes leading the switch from ovule integument into seed coat are still unknown. The single ovule integument of Ginkgo biloba acquires the typical characteristics of the seed coat long before the fertilization event. In this study, we investigated whether pollination triggers the transformation of the ovule integument into the seed coat. Transcriptomics and metabolomics analyses performed on ovules just prior and after pollination lead to the identification of changes occurring in Ginkgo ovules during this specific time. A morphological atlas describing the developmental stages of ovule development is presented. The metabolic pathways involved in the lignin biosynthesis and in the production of fatty acids are activated upon pollination, suggesting that the ovule integument starts its differentiation into a seed coat before the fertilization. Omics analyses allowed an accurate description of the main changes that occur in Ginkgo ovules during the pollination time frame, suggesting the crucial role of the pollen arrival on the progression of ovule development.

Taxonomic Re-Examination of Nine Rosellinia Types (Ascomycota, Xylariales) Stored in the Saccardo Mycological Collection.

In a recent monograph on the genus Rosellinia, type specimens worldwide were revised and re-classified using a morphological approach. Among them, some came from Pier Andrea Saccardo's fungarium stored in the Herbarium of the Padova Botanical Garden. In this work, we taxonomically re-examine via a morphological and molecular approach nine different Roselliniasensu Saccardo types. ITS1 and/or ITS2 sequences were successfully obtained applying Illumina MiSeq technology and phylogenetic analyses were carried out in order to elucidate their current taxonomic position. Only the ITS1 sequence was recovered for Rosellinia areolata, while for R. geophila, only the ITS2 sequence was recovered. We proposed here new combinations for Rosellinia chordicola, R. geophila and R. horridula, while for R. ambigua, R. areolata, R. australis, R. romana and R. somala, we did not suggest taxonomic changes compared to the current ones. The name Rosellinia subsimilis Sacc. is invalid, as it is a later homonym of R. subsimilis P. Karst. & Starbäck. Therefore, we introduced Coniochaeta dakotensis as a nomen novum for R. subsimilis Sacc. This is the first time that these types have been subjected to a molecular study. Our results demonstrate that old types are an important source of DNA sequence data for taxonomic re-examinations.

Evolution of isoprene emission in Arecaceae (palms).

Isoprene synthase (IspS) is the sole enzyme in plants responsible for the yearly emission in the atmosphere of thousands of tonnes of the natural hydrocarbon isoprene worldwide. Species of the monocotyledonous family Arecaceae (palms) are among the highest plant emitters, but to date no IspS gene from this family has been identified. Here, we screened with PTR-ToF-MS 18 genera of the Arecaceae for isoprene emission and found that the majority of the sampled species emits isoprene. Putative IspS genes from six different genera were sequenced and three of them were functionally characterized by heterologous overexpression in Arabidopsis thaliana, demonstrating that they encode functional IspS genes. Site-directed mutagenesis and expression in Arabidopsis demonstrated the functional relevance of a novel IspS diagnostic tetrad from Arecaceae, whose most variable amino acids could not preserve catalytic function when substituted by a putatively dicotyledonous-specific tetrad. In particular, mutation of threonine 479 likely impairs the open-closed transition of the enzyme by altering the network of hydrogen bonds between helices H1α, H, and I. These results shed new light on the evolution of IspS in monocots, suggesting that isoprene emission is an ancestral trait within the Arecaceae family. The identification of IspS from Arecaceae provides promising novel enzymes for the production of isoprene in heterologous systems and allows the screening and selection of commercially relevant palm varieties with lower environmental impact.

Arabidopsis Photosynthetic and Heterotrophic Cell Suspension Cultures.

Cell suspension cultures represent a widely used experimental tool suitable to perform a variety of structural and physiological studies in a more simplified system compared to the organism in toto. In this chapter we describe the methods routinely used in our laboratory to establish and maintain Arabidopsis photosynthetic and heterotrophic cell suspension cultures, containing either chloroplasts or amyloplasts, respectively. The use of these in vitro systems may allow to obtain insights into the unique features of chloroplasts versus non-green plastids, as well as their integration in the structural and metabolic compartmentalization of the plant cell.

FePO4 NPs Are an Efficient Nutritional Source for Plants: Combination of Nano-Material Properties and Metabolic Responses to Nutritional Deficiencies.

Phosphorous and iron are a macro- and micronutrient, respectively, whose low bioavailability can negatively affect crop productivity. There is ample evidence that the use of conventional P and Fe fertilizers has several environmental and economical disadvantages, but even though great expectations surround nanotechnology and its applications in the field of plant nutrition, little is known about the mechanisms underlying the uptake and use of these sub-micron particles (nanoparticles, NPs) by crop species. This work shows that cucumber and maize plants both use the nutrients borne by FePO4 NPs more efficiently than those supplied as bulk. However, morpho-physiological parameters and nutrient content analyses reveal that while cucumber plants (a Strategy I species with regard to Fe acquisition) mainly use these NPs as a source of P, maize (a Strategy II species) uses them preferentially for Fe. TEM analyses of cucumber root specimens revealed no cell internalization of the NPs. On the other hand, electron-dense nanometric structures were evident in proximity of the root epidermal cell walls of the NP-treated plants, which after ESEM/EDAX analyses can be reasonably identified as iron-oxyhydroxide. It appears that the nutritional interaction between roots and NPs is strongly influenced by species-specific metabolic responses.

The Rare Sugar Tagatose Differentially Inhibits the Growth of Phytophthora infestans and Phytophthora cinnamomi by Interfering With Mitochondrial Processes.

Rare sugars are monosaccharides with limited availability in nature and their biological functions are largely unknown. Among them, tagatose was developed as a low-calorie sweetener and showed beneficial effects on human health. Tagatose is metabolized by only certain microbial taxa and inhibits the growth of important crop pathogens (e.g., Phytophthora infestans), but its mode of action and the microbial responses are unknown. The aim of this study was to understand the tagatose mode of action against Phytophthora spp., with the final aim of developing new plant protection products. Tagatose inhibited P. infestans growth in vitro and caused severe ultrastructural alterations, with the formation of circular and concentric mitochondrial cristae. Decreased ATP content and reduced oxygen consumption rate (OCR) were found in tagatose-incubated P. infestans as compared to the control, with the consequent accumulation of reactive oxygen species (ROS) and induction of genes related to apoptosis and oxidative stress response. On the other hand, tagatose did not, or only slightly, affect the growth, cellular ultrastructure and mitochondrial processes in Phytophthora cinnamomi, indicating a species-specific response to this rare sugar. The mode of action of tagatose against P. infestans was mainly based on the inhibition of mitochondrial processes and this rare sugar seems to be a promising active substance for the further development of eco-friendly fungicides, thanks to its anti-nutritional properties on some phytopathogens and low risk for human health.

FePO4 nanoparticles produced by an industrially scalable continuous-flow method are an available form of P and Fe for cucumber and maize plants.

Nanomaterials are widely used in medical and pharmaceutical fields, but their application in plant nutrition is at its infancy. Phosphorous (P) and iron (Fe) are essential mineral nutrients limiting in a wide range of conditions the yield of crops. Phosphate and Fe fertilizers to-date on the market display low efficiency (P fertilizers) and low persistence in soil (Fe fertilizers) and negatively affect the environment. In the tentative to overcome these problems, we developed a continuous industrially scalable method to produce FePO4 NPs based on the rapid mixing of salt solutions in a mixing chamber. The process, that included the addition of citrate as capping agent allowed to obtain a stable suspension of NPs over the time. The NPs were tested for their effectiveness as P and Fe sources on two hydroponically grown crop species (cucumber and maize) comparing their effects to those exerted by non-nanometric FePO4 (bulk FePO4). The results showed that FePO4 NPs improved the availability of P and Fe, if compared to the non-nano counterpart, as demonstrated by leaf SPAD indexes, fresh biomasses and P and Fe contents in tissues. The results open a new avenue in the application of nanosized material in the field of plant nutrition and fertilization.

Plant biodiversity and regulation of photosynthesis in the natural environment.

MAIN CONCLUSION: Investigation of photosynthesis regulation in different plant groups exposed to variable conditions showed that all species have similar photosynthetic electron transport modulation while excess energy dissipation is species specific. Photosynthesis is regulated in response to dynamic environmental conditions to satisfy plant metabolic demands while also avoiding possible over-excitation of the electron transport chain and the generation of harmful reactive oxygen species. Photosynthetic organisms evolved several mechanisms to modulate light harvesting and electron transport efficiency to respond to conditions changing at different timescales, going from fast sun flecks to slow seasonal variations. These regulatory mechanisms changed during evolution of photosynthetic organisms, also adapting to various ecological niches, making the investigation of plant biodiversity highly valuable to uncover conserved traits and plasticity of photosynthetic regulation and complement studies on model species. In this work, a set of plants belonging to different genera of angiosperms, gymnosperms, ferns and lycophytes were investigated by monitoring their photosynthetic parameters in different seasons looking for common trends and differences. In all plants, analysed photosynthetic electron transport rate was found to be modulated by growth light intensity, ensuring a balance between available energy and photochemical capacity. Growth light also influenced the threshold where heat dissipation of excitation energy, a mechanism called non-photochemical quenching (NPQ), was activated. On the contrary, NPQ amplitude did not correlate with light intensity experienced by the plants but was a species-specific feature. The zeaxanthin-dependent component of NPQ, qZ, was found to be the most variable in different plants and its modulation influenced the intensity and the kinetic properties of the response.

Biocontrol traits of Bacillus licheniformis GL174, a culturable endophyte of Vitis vinifera cv. Glera.

BACKGROUND: Bacillus licheniformis GL174 is a culturable endophytic strain isolated from Vitis vinifera cultivar Glera, the grapevine mainly cultivated for the Prosecco wine production. This strain was previously demonstrated to possess some specific plant growth promoting traits but its endophytic attitude and its role in biocontrol was only partially explored. In this study, the potential biocontrol action of the strain was investigated in vitro and in vivo and, by genome sequence analyses, putative functions involved in biocontrol and plant-bacteria interaction were assessed. RESULTS: Firstly, to confirm the endophytic behavior of the strain, its ability to colonize grapevine tissues was demonstrated and its biocontrol properties were analyzed. Antagonism test results showed that the strain could reduce and inhibit the mycelium growth of diverse plant pathogens in vitro and in vivo. The strain was demonstrated to produce different molecules of the lipopeptide class; moreover, its genome was sequenced, and analysis of the sequences revealed the presence of many protein-coding genes involved in the biocontrol process, such as transporters, plant-cell lytic enzymes, siderophores and other secondary metabolites. CONCLUSIONS: This step-by-step analysis shows that Bacillus licheniformis GL174 may be a good biocontrol agent candidate, and describes some distinguished traits and possible key elements involved in this process. The use of this strain could potentially help grapevine plants to cope with pathogen attacks and reduce the amount of chemicals used in the vineyard.

The Hydrophobin HYTLO1 Secreted by the Biocontrol Fungus Trichoderma longibrachiatum Triggers a NAADP-Mediated Calcium Signalling Pathway in Lotus japonicus.

Trichoderma filamentous fungi are increasingly used as biocontrol agents and plant biostimulants. Growing evidence indicates that part of the beneficial effects is mediated by the activity of fungal metabolites on the plant host. We have investigated the mechanism of plant perception of HYTLO1, a hydrophobin abundantly secreted by Trichoderma longibrachiatum, which may play an important role in the early stages of the plant-fungus interaction. Aequorin-expressing Lotus japonicus suspension cell cultures responded to HYTLO1 with a rapid cytosolic Ca2+ increase that dissipated within 30 min, followed by the activation of the defence-related genes MPK3, WRK33, and CP450. The Ca2+-dependence of these gene expression was demonstrated by using the extracellular Ca2+ chelator EGTA and Ned-19, a potent inhibitor of the nicotinic acid adenine dinucleotide phosphate (NAADP) receptor in animal cells, which effectively blocked the HYTLO1-induced Ca2+ elevation. Immunocytochemical analyses showed the localization of the fungal hydrophobin at the plant cell surface, where it forms a protein film covering the plant cell wall. Our data demonstrate the Ca2+-mediated perception by plant cells of a key metabolite secreted by a biocontrol fungus, and provide the first evidence of the involvement of NAADP-gated Ca2+ release in a signalling pathway triggered by a biotic stimulus.

Chloroplast Ca2+ Fluxes into and across Thylakoids Revealed by Thylakoid-Targeted Aequorin Probes.

Chloroplasts require a fine-tuned control of their internal Ca2+ concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca2+ signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca2+ homeostasis and in the modulation of chloroplast Ca2+ signals in vivo, we targeted the bioluminescent Ca2+ reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (Arabidopsis thaliana). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca2+ levels in the thylakoid lumen were maintained at about 0.5 µm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca2+ dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca2+ signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca2+ changes in the thylakoid lumen. Localized Ca2+ increases also were observed on the thylakoid membrane surface, mirroring transient Ca2+ changes observed for the bulk stroma, but with specific Ca2+ dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca2+ changes, suggesting a new scenario for light-to-dark-induced Ca2+ fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca2+ storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca2+ signaling networks within chloroplasts and plant cells.

A Rapid and Efficient Method to Obtain Photosynthetic Cell Suspension Cultures of Arabidopsis thaliana.

Photosynthetic cell suspension cultures are a useful experimental system to analyze a variety of physiological processes, bypassing the structural complexity of the plant organism in toto. Nevertheless, cell cultures containing functional chloroplasts are quite difficult to obtain, and this process is usually laborious and time-consuming. In this work a novel and rapid method to set up photosynthetic cell suspension cultures from the model plant Arabidopsis thaliana was developed. The direct germination of Arabidopsis seeds on a sucrose-containing agarized culture medium supplemented with 0.25 µg/ml 6-benzylaminopurine and 0.5 µg/ml 2,4-dichlorophenoxyacetic acid caused the straightforward formation of green calli at the level of seedling hypocotyls. The subsequent transfer of these calli in liquid culture medium containing the same concentrations of phytohormones and gradually decreasing sucrose levels allowed for the establishment of chloroplast-containing cell suspension cultures, containing functional chloroplasts, in a much faster way than previously described procedures. Pulse amplitude modulation analyses, measurements of oxygen evolution and electron transport rate, together with confocal and electron microscopy observations, confirmed the photosynthetic efficiency of these cell suspension cultures. The described procedure lends itself as a simple and effective way to obtain a convenient tool for a wide array of structural and functional studies on chloroplasts.

Endocytic pathways involved in PLGA nanoparticle uptake by grapevine cells and role of cell wall and membrane in size selection.

KEY MESSAGE: PLGA NPs' cell uptake involves different endocytic pathways. Clathrin-independent endocytosis is the main internalization route. The cell wall plays a more prominent role than the plasma membrane in NPs' size selection. In the last years, many studies on absorption and cell uptake of nanoparticles by plants have been conducted, but the understanding of the internalization mechanisms is still largely unknown. In this study, polydispersed and monodispersed poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs) were synthesized, and a strategy combining the use of transmission electron microscopy (TEM), confocal analysis, fluorescently labeled PLGA NPs, a probe for endocytic vesicles (FM4-64), and endocytosis inhibitors (i.e., wortmannin, ikarugamycin, and salicylic acid) was employed to shed light on PLGA NP cell uptake in grapevine cultured cells and to assess the role of the cell wall and plasma membrane in size selection of PLGA NPs. The ability of PLGA NPs to cross the cell wall and membrane was confirmed by TEM and fluorescence microscopy. A strong adhesion of PLGA NPs to the outer side of the cell wall was observed, presumably due to electrostatic interactions. Confocal microscopy and treatment with endocytosis inhibitors suggested the involvement of both clathrin-dependent and clathrin-independent endocytosis in cell uptake of PLGA NPs and the latter appeared to be the main internalization pathway. Experiments on grapevine protoplasts revealed that the cell wall plays a more prominent role than the plasma membrane in size selection of PLGA NPs. While the cell wall prevents the uptake of PLGA NPs with diameters over 50 nm, the plasma membrane can be crossed by PLGA NPs with a diameter of 500-600 nm.

Impact of Phenylpropanoid Compounds on Heat Stress Tolerance in Carrot Cell Cultures.

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on projection to latent structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton.