Malaria is one of the most severe health problems facing the world today. Until the mid-twentieth century, Europe was an endemic area of malaria, with the Balkan countries being heavily infested. Sibling species belonging to the Anopheles maculipennis complex are well-known as effective vectors of Plasmodium in Europe. A vast number of human malaria cases in the past in the former Yugoslavia territory have stressed the significance of An. maculipennis complex species as primary and secondary vectors. Therefore, the present study evaluates the species composition, geographic distribution and abundance of these malaria vector species. Mosquitoes were collected in the northern Serbian province of Vojvodina and analysed by PCR-RFLP, multiplex PCR and sequencing of the ITS2 intron of genomic rDNA. Four sibling species of the An. maculipennis complex were identified. Both larvae and adults of the recently described species An. daciae were identified for the first time in Serbia. In 250 larval samples, 109 (44%) An. messeae, 90 (36%) An. maculipennis s.s., 33 (13%) An. daciae and 18 (7%) An. atroparvus were identified. In adult collections, 81 (47%) An. messeae, 55 (32%) An. daciae, 33 (19%) An. maculipennis s.s., and 3 (2%) An. atroparvus were recorded. The most abundant species in Vojvodina was An. messeae, whereas An. atroparvus was confirmed a rare species in all parts. Since this species is a potentially, highly competent malarial vector, low population density could be crucial to prevent a new establishment of endemic malaria transmission in Serbia.

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Plants produce a wide variety of secondary metabolites, which often are of interest to pharmaceutical and nutraceutical industry. Plant-cell cultures allow producing these metabolites in a standardised manner, independently from various biotic and abiotic factors difficult to control during conventional cultivation. However, plant-cell fermentation proves to be very difficult, since these chemically complex compounds often result from the interaction of different biosynthetic pathways operating in different cell types. To simulate such interactions in cultured cells is a challenge. Here, we present a microfluidic bioreactor for plant-cell cultivation to mimic the cell–cell interactions occurring in real plant tissues. In a modular set-up of several microfluidic bioreactors, different cell types can connect through a flow that transports signals or metabolites from module to module. The fabrication of the chip includes hot embossing of a polycarbonate housing and subsequent integration of a porous membrane and in-plane tube fittings in a two-step ultrasonic welding process. The resulting microfluidic chip is biocompatible and transparent. Simulation of mass transfer for the nutrient sucrose predicts a sufficient nutrient supply through the membrane. We demonstrate the potential of this chip for plant cell biology in three proof-of-concept applications. First, we use the chip to show that tobacco BY-2 cells in suspension divide depending on a “quorum-sensing factor” secreted by proliferating cells. Second, we show that a combination of two Catharanthus roseus cell strains with complementary metabolic potency allows obtaining vindoline, a precursor of the anti-tumour compound vincristine. Third, we extend the approach to operationalise secretion of phytotoxins by the fungus Neofusicoccum parvum as a step towards systems to screen for interorganismal chemical signalling.

Salinity is a serious challenge to global agriculture and threatens human food security. plant cells can respond to salt stress either by activation of adaptive responses, or by programmed cell death. the mechanisms deciding the respective response are far from understood, but seem to depend on the degree, to which mitochondria can maintain oxidative homeostasis. Using plant peptoQ, a Trojan Peptoid, as vehicle, it is possible to transport a coenzyme Q10 (CoQ10) derivative into plant mitochondria. We show that salinity stress in tobacco BY-2 cells (Nicotiana tabacum L. cv Bright Yellow-2) can be mitigated by pretreatment with plant PeptoQ with respect to numerous aspects including proliferation, expansion, redox homeostasis, and programmed cell death. We tested the salinity response for transcripts from nine salt-stress related-genes representing different adaptive responses. While most did not show any significant response, the salt response of the transcription factor NtNAC, probably involved in mitochondrial retrograde signaling, was significantly modulated by the plant peptoQ. Most strikingly, transcripts for the mitochondrial, Mn-dependent Superoxide Dismutase were rapidly and drastically upregulated in presence of the peptoid, and this response was disappearing in presence of salt. the same pattern, albeit at lower amplitude, was seen for the sodium exporter SOS1. The findings are discussed by a model, where plant PeptoQ modulates retrograde signalling to the nucleus leading to a strong expression of mitochondrial SoD, what renders mitochondria more resilient to perturbations of oxidative balance, such that cells escape salt induced cell death and remain viable. By the year 2050, world agriculture should be able to boost production of food crops by 70% to feed the projected 9.1 billion people 1. Salt stress is one of the major challenges to this effort and has already affected 20% of cultivated land worldwide. Global warming and suboptimal irrigation accentuate the problem 2,3. The molecular, cellular, and physiological mechanisms underlying the severe effect of salinity on plant growth, development and yield have been reviewed comprehensively 3-5. Based on their response to salt stress, plants are categorised into the tolerant halophytes and the sensitive glycophytes. Unfortunately, most of the crop species belong to the

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question: how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seed embryos of Catharanthus roseus were found to differ not only morphologically, but also in their metabolic competence. This differential competence became manifest not only under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA-elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not “de-differentiated”, but can differ in metabolic competence. In addition to elicitation and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.

Key message Paclitaxel synthesis in Taxus cells correlates with a cell-fate switch that leads to vacuoles of a glossy appearance and vermiform mitochondria. This switch depends on actin and apoplastic respiratory burst. Abstract Plant cell fermentation, the production of valuable products in plant cell culture, has great potential as sustainable alternative to the exploitation of natural resources for compounds of pharmaceutical interest. However, the success of this approach has remained limited, because the cellular aspects of metabolic competence are mostly unknown. The production of the anti-cancer alkaloid Paclitaxel has been, so far, the most successful case for this approach. In the current work, we map cellular aspects of alkaloid synthesis in cells of Taxus chinensis using a combination of live-cell imaging, quantitative physiology, and metabolite analysis. We show evidence that metabolic potency correlates with a differentiation event giving rise to cells with large vacuoles with a tonoplast that is of a glossy appearance, agglomerations of lipophilic compounds, and multivesicular bodies that fuse with the plasma membrane. Cellular features of these glossy cells are bundled actin, more numerous peroxisomes, and vermiform mitochondria. The incidence of glossy cells can be increased by aluminium ions, and this increase is significantly reduced by the actin inhibitor Latrunculin B, and by diphenylene iodonium, a specific inhibitor of the NADPH oxidase Respiratory burst oxidase Homologue (RboH). It is also reduced by the artificial auxin Picloram. This cellular fingerprint matches the implications of a model, where the differentiation into the glossy cell type is regulated by the actin–auxin oscillator that in plant cells acts as dynamic switch between growth and defence.

Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs). Due to their low abundance in plant tissues at a simultaneously high demand, modes of production alternative to conventional extraction are mandatory. Plant cell fermentation might become one of these alternatives, yet decades of research have shown limited success to certain product classes, leading to the question, how to preserve the intrinsic ability to produce TIAs (metabolic competence) in cell culture? We used the strategy to use the developmental potency of mature embryos to generate such strains. Two cell strains (C1and C4) from seeds of Catharanthus roseus were found not only to differ morphologically, but also in their metabolic competence. This differential competence became manifest under phytohormone elicitation, but also upon feeding with alkaloid pathway precursors. The more active strain C4 formed larger cell aggregates and was endowed with longer mitochondria. These cellular features were accompanied by higher alkaloid accumulation in response to methyl jasmonate (MeJA) elicitation. The levels of catharanthine could be increased significantly, while the concurrent vindoline branch of the pathway was blocked, such that no bisindole alkaloids were detectable. By feeding vindoline to MeJA elicited C4 cells, vincristine became detectable; however, only to marginal amounts. In conclusion, these results show that cultured cells are not just “de-differentiated”, but can differ in metabolic competence. In addition to elicitation, and precursor feeding, the cellular properties of the “biomatter” are highly relevant for the success of plant cell fermentation.