This chapter presents a holistic and quantitative approach to the carbon cycle in plant systems biology. It includes (rapid) phenotyping and monitoring of physiological key interactions of plants with its respective soil and atmospheric environment (soil plant atmospheric continuum-SPAC). The approach aims at qualifying and quantifying key components of this microhabitat as influenced by a single plant or a local group of plants in order to contribute to a flux-based modelling approach. The toolset consists of plant biometry, gas exchange, metabolomics, ionomics, root exudate characterization as well as soil biological and physical-chemical characterization. The results are presented as a basic interaction and input-output model aka conceptual system model employing H. T. Odum-style plots based on empirical data.

The large structural diversity of specialized metabolites represents a substantial challenge in untargeted metabolomics. Modern LC–QTOF instruments can provide three- to four-digit numbers of auto-MS/MS spectra from sample sets. This case study utilizes twelve structurally closely related flavonol glycosides, characteristic specialized metabolites of plant tissues, some of them isomeric and isobaric, to illustrate the possibilities and limitations of their identification. This process requires specific software tools that perform peak picking and feature alignment after spectral deconvolution and facilitate molecular structure base searching with subsequent in silico fragmentation to obtain initial ideas about possible structures. The final assignment of a putative identification, so long as spectral databases are not complete enough, requires structure searches in a chemical reference database, such as SciFindern, in attempts to obtain additional information about specific product ions of a metabolite candidate or check its feasibility. The highlighted problems in this process not only apply to specialized metabolites in plants but to those occurring in other organisms as well. This case study is aimed at providing guidelines for all researchers who obtain data from such analyses but are interested in deeper information than just Venn diagrams of the feature distribution in their sample groups.

Polyploidy has substantially contributed to successful plant evolution, and is often connected to a higher resilience to environmental stress. We test the hypothesis that polyploids tolerate light stress better than diploids. The Ranunculus auricomus complex comprises diploid (2x), tetraploid (4x), and hexaploid (6x) cytotypes, the former of which occur in shaded habitats and the latter more in open, sun-exposed habitats in Central Europe. In this study, we experimentally explored the effects of ploidy and photoperiod extension on the efficiency of photosystem II in the three cytotypes in climate growth chambers. Quantum yields and various coefficients that can be calculated from light curve, Kautsky curve, and fluorescent transient OJIP experiments provided support for the hypothesis that, in comparison to diploids, the improved regulation of excess light by more efficient photochemical and non-chemical quenching in polyploids might have facilitated the adaptation to unshaded habitats. We suggest how lower stress levels in reproductive tissues of polyploids might have favored asexual reproduction.

The evolutionary advantage of different sexual systems in multicellular eukaryotes is still not well understood, because the differentiation into male and female individuals halves offspring production compared with asexuality. Here we propose that various physiological adaptations to oxidative stress could have forged sessility versus motility, and consequently the evolution of sexual systems in multicellular animals, plants, and fungi. Photosynthesis causes substantial amounts of oxidative stress in photoautotrophic plants and, likewise, oxidative chemistry of polymer breakdown, cellulose and lignin, for saprotrophic fungi. In both cases, its extent precludes motility, an additional source of oxidative stress. Sessile life form and the lack of neuronal systems, however, limit options for mate recognition and adult sexual selection, resulting in inefficient mate-searching systems. Hence, sessility requires that all individuals can produce offspring, which is achieved by hermaphroditism in plants and/or by multiple mating types in fungi. In animals, motility requires neuronal systems, and muscle activity, both of which are highly sensitive to oxidative damage. As a consequence, motility has evolved in animals as heterotrophic organisms that (1) are not photosynthetically active, and (2) are not primary decomposers. Adaptations to motility provide prerequisites for an active mating behavior and efficient mate-searching systems. These benefits compensate for the “cost of males”, and may explain the early evolution of sex chromosomes in metazoans. We conclude that different sexual systems evolved under the indirect physiological constraints of lifestyles.

Flavonoids are rather common plant phenolic constituents that are known for potent antioxidant effects and can be beneficial for human health. Flavonoids with a pyrogallol moiety are highly efficient reducing agents with possible pro- and antioxidant effects, depending on the reaction milieu. Therefore, the redox properties of myricetin and tricetin were investigated by differential pulse voltammetry and deoxyribose degradation assay. Tricetin proved to be a good antioxidant but only showed negligible pro-oxidant activity in one of the deoxyribose degradation assay variants. Compared to tricetin, myricetin showed pro- and antioxidant effects. The more efficient reducing properties of myricetin are probably caused by the positive mesomeric effect of the enolic 3-hydroxy group on ring C. It is evident that the antioxidant properties of structurally similar flavonoids can be converted to apparent pro-oxidant effects by relatively small structural changes, such as hydroxylation. Since reactive oxygen species (ROS) often serve as secondary messengers in pathological and physiological processes in animal and plant cells, the pro- and antioxidant properties of flavonoids are an important part of controlling mechanisms of tissue signal cascades.

Reactive oxygen species (ROS) are known for their participation in various physiological and pathological processes in organisms, including ageing or degeneration. Kynurenine pathway metabolites, such as kynurenic (KYNA) or xanthurenic (XA) acid, can affect neurodegenerative diseases due to their ROS scavenging and Fe ion coordination complex formation but insights are still incomplete. Therefore, we investigated the formation and antioxidant capabilities of KYNA– and XA–Fe complexes by nano-electrospray−mass spectrometry, differential pulse voltammetry, deoxyribose degradation and FeII autoxidation assays. XA formed coordination complexes with FeII or FeIII ions and was an effective antioxidant. By contrast, only FeII–KYNA complexes could be detected. Moreover, KYNA showed no antioxidant effects in the FeCl3/ascorbic acid deoxyribose degradation assay variant and only negligible activities in the FeII autoxidation assay. Coordination complexes of Fe ions with KYNA probably stabilize KYNA in its keto tautomer form. Nevertheless, both KYNA and XA exhibited sufficient antioxidant activities in some of the employed assay variants. The results provide evidence that both have the potential to alleviate neurodegenerative diseases by helping to maintain tissue redox homeodynamics.

The volume and complexity of commercial bioreactors for sterile hydroponics and hairy roots are too large for comparative analysis of many cultures. Here a small-scale bioreactor fabricated from standard glass materials and suitable for both airlift and bubble aeration mode is described. The performance of the bioreactor was tested by growing oilseed rape (Brassica napus L.) and rose plants (Rosa canina L.) in sterile hydroponics and by cultivating hairy roots of henbane (Hyoscyamus niger L.) and sesame (Hyoscyamus niger L.). Plants grown in hydroponics for up to six weeks did not show chloroses or necroses. Hairy roots grew faster or comparably fast in bioreactors as compared to shaking flasks. Root exudates of roses and exudates of hairy roots of henbane were subjected to targeted and nontargeted analysis by HPLC coupled with optical and mass spectrometric detectors. The diversity and concentration of hairy root exudates were higher in bioreactors than in shaking flasks. The composition of hairy root exudates of three accessions of H. niger did not match the genetic relatedness among the accessions. Hairy roots of Hyoscyamus niger exuded salicylic acid in amounts varying among plant accessions and between bioreactors and shaking flask cultures.

Background: The alkaloid 8-hydroxyquinoline (8HQ) is well-known for various biological activities, including antioxidant effects and especially for the formation of coordination complexes with various transition metals, such as iron, amongst others. Therefore, 8HQ was extensively explored as a promising antineurodegenerative agent. However, other authors noted pro-oxidant effects of 8HQ. Here, we explore the pro- and antioxidant properties of 8HQ, especially in context of coordination complexes with iron (II) and iron (III). Methods: Nano-electrospray−mass spectrometry, differential pulse voltammetry, deoxyribose degradation, iron (II) autoxidation, and brine shrimp mortality assays were used. Results: 8HQ formed a complex mixture of coordination complexes with iron (II) and iron (III). Furthermore, 8HQ showed antioxidant effects but no pro-oxidant ones. In the brine shrimp mortality assay, 8HQ demonstrated toxicity that decreased in the presence of iron (III). Conclusions: 8HQ is a potent antioxidant whose effects depend not only on the formation of the coordination complexes with iron ions, but surely on the scavenging activities due to the redox properties of the 8-hydroxyl group. No pro-oxidant effects were observed in the set of the used assays.

The African pepper bark tree, Warburgia ugandensis, accumulates antimicrobial drimane sesquiterpenes in all of its organs. One hypothesis states that plant defense compounds determine endophyte community structure. Another hypothesis suggests that they just facilitate the endophytic lifestyle by exerting a balanced antagonism. To explore this, a representative selection of endophytic bacterial and fungal isolates from this tree species was assayed together with six non-endophytic strains to determine their tolerance and susceptibility to the root and leaf extract fraction containing high and low drimane sesquiterpene amounts respectively. Inhibitory effects were explored by assessing both growth and growth efficiency, the latter of which relates respiratory activity to growth. The susceptibility of the tested strains showed considerable variation and the obtained patterns did not allow a clear distinction between root and leaf endophytes as well as endophytes and non-endophytes. In addition, all strains were also assayed against juglone, an antimicrobial and redox-active aromatic naphthoquinone. A comparison of differential pulse voltammograms and efficacy in variants of the deoxyribose degradation assay revealed that drimane sesquiterpenes possess anti- and pro-oxidant activities that compare to those of juglone. Leaf endophytes showed higher resistance to oxidative stress than root endophytes, quite contrary to the actual exposure. The obtained results support the notion that structural diverse plant defense compounds can contribute to a balanced antagonism against but not to structuring of endophyte communities. Oxidative stress seems to be involved in generating this effect albeit it cannot explain it alone.

Meiosis, the key step of sexual reproduction, persists in facultative apomictic plants functional to some extent. However, it still remains unclear how and why proportions of reproductive pathways vary under different environmental stress conditions. We hypothesized that oxidative stress mediates alterations of developmental pathways. In apomictic plants we expected that megasporogenesis, the stage directly after meiosis, would be more affected than later stages of seed development. To simulate moderate stress conditions we subjected clone-mates of facultative apomictic Ranunculus auricomus to 10 h photoperiods, reflecting natural conditions, and extended ones (16.5 h). Reproduction mode was screened directly after megasporogenesis (microscope) and at seed stage (flow cytometric seed screening). Targeted metabolite profiles were performed with HPLC-DAD to explore if and which metabolic reprogramming was caused by the extended photoperiod. Prolonged photoperiods resulted in increased frequencies of sexual vs. aposporous initials directly after meiosis, but did not affect frequencies of sexual vs. asexual seed formation. Changes in secondary metabolite profiles under extended photoperiods affected all classes of compounds, and c. 20% of these changes separated the two treatments. Unexpectedly, the renowned antioxidant phenylpropanoids and flavonoids added more to clone-mate variation than to treatment differentiation. Among others, chlorophyll degradation products, non-assigned phenolic compounds and more lipophilic metabolites also contributed to the dissimilarity of the metabolic profiles of plants that had been exposed to the two different photoperiods. The hypothesis of moderate light stress effects was supported by increased proportions of sexual megaspore development at the expense of aposporous initial formation. The lack of effects at the seed stage confirms the basic assumption that only meiosis and sporogenesis would be sensitive to light stress. The concomitant change of secondary metabolite profiles, as a systemic response at this early developmental stage, supports the notion that oxidative stress could have affected megasporogenesis by causing the observed metabolic reprogramming. Hypotheses of genotype-specific responses to prolonged photoperiods are rejected.

The flavanol (±)-catechin shows an OH group but no 4-keto group on ring C (C3), and no conjugation between ring A and B. The related flavanone (+)-eriodictyol has a keto group on C4 but no 3-OH group on ring C. (+)-Taxifolin, another flavanone, has an OH on C3 and a keto group on C4 of the C ring. Deoxyribose degradation assay systems, with hydrogen peroxide and ascorbic acid either added or omitted, were performed in variants in which Fe(III) was added in a complex with ethylenediaminetetraacetic acid (EDTA). In combination with differential pulse voltammetry (DVP), the specific redox-chemical contributions of the ring A m-dihydroxyl groups could be explored more specifically in addition to those of the traditionally investigated o-dihydroxyl groups of ring B.

Disposal of organic plant wastes and by-products from the food or pharmaceutical industries usually involves high costs. In the present study, 42 samples derived from such by-products were screened in vitro against Cryptosporidium parvum, a protozoan parasite that may contaminate drinking water and cause diarrhoea. The novel bioassay was previously established in the microtitre plate format. Human ileocaecal adenocarcinoma (HCT-8) cell cultures were seeded with C. parvum oocysts and parasite development was monitored by an indirect fluorescent antibody technique (IFAT) and microscopic assessment for clusters of secondary infection (CSI). Minimum inhibitory concentrations (MICs) and potential detrimental effects on the host cells were determined. An ethanolic extract from olive (Olea europaea) pomace, after oil pressing and phenol recovery, reproducibly inhibited C. parvum development (MIC = 250–500 μg mL−1, IC50 = 361 (279–438) μg mL−1, IC90 = 467 (398–615) μg mL−1). Accordingly, tyrosol, hydroxytyrosol, trans-coniferyl alcohol and oleuropein were selected as reference test compounds, but their contributions to the observed activity of the olive pomace extract were insignificant. The established test system proved to be a fast and efficient assay for identifying anti-cryptosporidial activities in biological waste material and comparison with selected reference compounds.

The alkaloid 8-hydroxyquinoline (HQ) was pointed out as potential phytosiderophore that is taken up unspecifically by iron acquisition strategy I and II plants. HQ in root exudate of the knapweed Centaurea diffusa even was considered as a factor contributing to the invasive success of this species. This study compares the iron supply efficiencies of the Fe-HQ complex and Fe-EDTA in hydroponic cultures of rapeseed (Brassica napus) as a plant model system to explore the proposed function. Iron (FeCl3) was supplied in 2 and 10 mu M concentrations in a 1:1 ratio with the ligand (HQ). After 20 days, Fe content, lipid peroxidation, superoxide anion, hydrogen peroxide, and ascorbic acid concentrations, various enzyme activities associated with antioxidant defences and programmed cell death (PCD), and nuclear condensation were determined. Iron supply in the form of a Fe-HQ complex clearly was less efficient. These plants developed chlorosis and showed symptoms of non-autolytic PCD similar to those that had been subjected to Fe deprivation. Reactive oxygen species (ROS) concentration levels and enzyme activities (superoxide dismutase, catalase, alkaline protease, caspase-3-like and deoxyribonuclease) resembled more those observed in plants which suffered from Fe deprivation, than those of Fe-sufficient Fe-EDTA supplied plants. The results do not support the putative phytosiderophore function alleged to HQ in the studied concentration range (2-10 mu M) but instead corroborate the one hypothesis explaining HQ toxicity: Fe in complex with HQ is not released efficiently enough from the complex to be available for metalloenzymes that require it as a co-factor. (C) 2014 Elsevier B.V. All rights reserved.

Low-molecular-weight metabolites (LMWMs) comprise primary or central and a plethora of intermediary or secondary metabolites, all of which are characterized by a molecular weight below 900 Dalton. The latter are especially prominent in sessile higher organisms, such as plants, corals, sponges and fungi, but are produced by all types of microbial organisms too. Common to all of these carbon molecules are oxygen, nitrogen and, to a lesser extent, sulfur, as heteroatoms. The latter can contribute as electron donators or acceptors to cellular redox chemistry and define the potential of the molecule to enter charge-transfer complexes. Furthermore, they allow LMWMs to serve as organic ligands in coordination complexes of various inorganic metals as central atoms. Especially the transition metals Fe, Cu and Mn can catalyze one electron reduction of molecular oxygen, which results in formation of free radical species and reactive follow-up reaction products. As antioxidants LMWMs can scavenge free radicals. Depending on the chemical environment, the same LMWMs can act as pro-oxidants by reducing molecular oxygen. The cellular regulation of redox homeostasis, a balance between oxidation and reduction, is still far from being understood. Charge-transfer and coordination complex formation with metals shapes LMWMs into gel-like matrices in the cytosol. The quasi-polymer structure is lost usually during the isolation procedure. In the gel state, LMWMs possess semiconductor properties. Also proteins and membranes are semiconductors. Together they can represent biotransistor components that can be part of a chemoelectrical signaling system that coordinates systems chemistry by initiating cell differentiation or tissue homeostasis, the activated and the resting cell state, when it is required. This concept is not new and dates back to Albert Szent-Gyorgyi.

Anthranilic acid (ANA) and 3-hydroxyanthranilic acid (3-HANA) are kynurenine pathway intermediates of the tryptophan metabolism. A hitherto unemployed method combination, differential pulse voltammetry, mass spectrometry (nano-ESI-MS), deoxyribose degradation and iron(II) autoxidation assays has been employed for studying of their redox chemistry and their interactions with iron(II) and iron(III) ions. Both acids inhibited the Fenton reaction by iron chelation and ROS scavenging in the deoxyribose degradation assay. In the iron(II) autoxidation assay, anthranilic acid showed antioxidant effects, whereas 3-hydroxyanthranilic acid exhibited apparent pro-oxidant activity. The differential pulse voltammograms of free metabolites and their iron(II) coordination complexes reflected these properties. Nano-ESI-MS confirmed ANA and 3-HANA as efficient iron(II) chelators, both of which form coordination complexes of ligand:iron(II) ratio 1:1, 2:1, and 3:1. In addition, nano-ESI-MS analyses of the oxidation effects by hydroxyl radical attack identified 3-HANA as strikingly more susceptible than ANA. 3-HANA susceptibility to oxidation may explain its decreased concentrations in the reaction mixture. The presented observations can add to explaining why 3-HANA levels decrease in patients with some neurological and other diseases which can often associated with elevated concentrations of ROS.

Low-molecular-weight metabolites (LMWMs) comprise primary or central and a plethora of intermediary or secondary metabolites, all of which are characterized by a molecular weight below 900 Dalton. The latter are especially prominent in sessile higher organisms, such as plants, corals, sponges and fungi, but are produced by all types of microbial organisms too. Common to all of these carbon molecules are oxygen, nitrogen and, to a lesser extent, sulfur, as heteroatoms. The latter can contribute as electron donators or acceptors to cellular redox chemistry and define the potential of the molecule to enter charge-transfer complexes. Furthermore, they allow LMWMs to serve as organic ligands in coordination complexes with various inorganic metals as central atoms. Especially the transition metals Fe, Cu, and Mn can catalyze one electron reduction of molecular oxygen, which results in formation of free radical species and reactive follow-up reaction products. As antioxidants LMWMs can scavenge free radicals. Depending on the chemical environment, the same LMWMs can act as pro-oxidants by reducing molecular oxygen. The cellular regulation of redox homeostasis, a balance between oxidation and reduction, is still far from being understood. Charge-transfer and coordination complex formation with metals shapes LMWMs into gel-like matrices in the cytosol. The quasi-polymer structure is lost usually during the isolation procedure. In the gel state, LMWMs possess semiconductor properties. Also proteins and membranes are semiconductors. Together they can represent biotransistor components that can be part of a chemoelectrical signaling system that coordinates systems chemistry by initiating cell differentiation or tissue homeostasis, the activated and the resting cell state, when it is required. This concept is not new and dates back to Albert Szent-Gyorgyi.

The tryptophan metabolite, quinolinic (2,3-pyridinedicarboxylic) acid, is known as an endogenous neurotoxin. Quinolinic acid can form coordination complexes with iron or copper. The effects of quinolinic acid on reactive oxygen species production in the presence of iron or copper were explored by a combination of chemical assays, classical site-specific and ascorbic acid-free variants of the deoxyribose degradation assay, and mass spectrometry (ESI-MS). Quinolinic acid showed evident antioxidant activity in chemical assays, but the effect was more pronounced in the presence of copper as transition metal catalyst than in presence of iron. Nano-ESI-MS confirmed the ability of quinolinic acid to form coordination complexes with iron(II) or copper(II) and quinolinic acid stability against oxidative attack by hydroxyl radicals. The results illustrate a highly milieu-dependent quinolinic acid chemistry when it enters reactions as competitive ligand.