Marco Vervliet-Scheebaum

Metabolite profiling of the moss Physcomitrella patens reveals evolutionary conservation of osmoprotective substances

The moss Physcomitrella patens is suitable for systems biology studies, as it can be grown axenically under standardised conditions in plain mineral medium and comprises only few cell types. We report on metabolite profiling of two major P. patens tissues, filamentous protonema and leafy gametophores, from different culture conditions. A total of 96 compounds were detected, 21 of them as yet unknown in public databases. Protonema and gametophores had distinct metabolic profiles, especially with regard to saccharides, sugar derivates, amino acids, lignin precursors and nitrogen-rich storage compounds. A hydroponic culture was established for P. patens, and was used to apply drought stress under physiological conditions. This treatment led to accumulation of osmoprotectants, such as altrose, maltitol, ascorbic acid and proline. Thus, these osmoprotectants are not unique to seed plants but have evolved at an early phase of the colonization of land by plants.

Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division, Chloroplast Shaping, Cell Patterning, Plant Development, and Gravity Sensing

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in P. patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss P. patens.

Evaluating the necessity of additional aquatic plant testing by comparing the sensitivities of different species

At present, at least three and up to five plant species are required to assess the potential risks of herbicides to non-target aquatic plants. Several regulatory authorities are considering whether there should be further requirements based on concerns about the possible selectivity of herbicides (e.g., specific modes of action against dicotyledonous plants). The relative sensitivity of a range of aquatic plants is assessed in our work in order to evaluate the implications of differences in species sensitivity for aquatic risk assessment of herbicides. We therefore present results from ecotoxicological tests performed at Syngenta Crop Protection AG on various aquatic plants and compare them to available studies and results in literature. The criterion used for sensitivity ranking is the EC50 (median effect concentration) value, which allows a better comparison of values from different testing methods and conditions. The overall results obtained in the present work show that the aquatic risk assessment procedure for herbicides based on Lemna sp. and algae is sufficiently protective while identifying potential toxicity to non-target plants. Only few exceptions concerning herbicides with selective modes of action (e.g., auxin simulators) may require additional species testing for proper risk assessment.

A uniquely high number of ftsZ genes in the moss Physcomitrella patens

Plant FtsZ proteins are encoded by two small nuclear gene families (FtsZ1 and FtsZ2) and are involved in chloroplast division. From the moss Physcomitrella patens, four FtsZ proteins, two in each nuclear gene family, have been characterised and described so far. In the recently sequenced P. patens genome, we have now found a fifth ftsZ gene. This novel gene has a genomic structure similar to PpftsZ1-1. According to phylogenetic analysis, the encoded protein is a member of the FtsZ1 family, while PpFtsZ1-2, together with an orthologue from Selaginella moellendorffii, forms a separate clade. Further, this new gene is expressed in different gametophytic tissues and the encoded protein forms filamentous networks in chloroplasts, is found in stromules, and acts in plastid division. Based on all these results, we have renamed the PpFtsZ proteins of family 1 and suggest the existence of a third FtsZ family. No species is known to encode more FtsZ proteins per haploid genome than P. patens.

A microcosm system to evaluate the toxicity of the triazine herbicide simazine on aquatic macrophytes.

This study evaluates the effects of the triazine herbicide simazine in an outdoor pond microcosm test system that contained two submerged rooted species (Myriophyllum spicatum and Elodea canadensis) and two emergent rooted species (Persicaria amphibia and Glyceria maxima) over a period of 84 days. Simazine was applied to the microcosms at nominal concentrations of 0.05, 0.5 and 5 mg/L. General biological endpoints and physiological endpoints were used to evaluate herbicide toxicity on macrophytes and the algae developing naturally in the system. Concentration-related responses of macrophytes and algae were obtained for the endpoints selected, resulting in a no observed ecologically adverse effect concentration (NOEAEC) at simazine concentrations of 0.05 mg active ingredient/L after 84 days. E. canadensis was the most negatively affected species based on length increase, which was consistently a very sensitive parameter for all macrophytes. The experimental design presented might constitute a suitable alternative to conventional laboratory single-species testing.

Metabolic control of transcriptional-translational control loops (TTCL) by circadian oscillations in the redox- and phosphorylation state of cells

Abstract Evolution from prokaryotic to eukaryotic organisms was paralleled by a corresponding evolution in energy metabolism. From primeval fermentation, energy conservation progressed to anaerobic photosynthesis and then to carbon dioxide fixation with acceptance of electrons by water and the evolution of oxygen. In a progressively oxygenic biosphere, respiration developed with oxygen as a terminal electron acceptor. Evolving life was paralleled by a corresponding evolution of tropospheric O2/CO2 composition and the feedback of oxygen on life processes via reactive oxygen and reactive nitrogen species, which as signalling molecules became crucial for the control of development of pro- and eukaryotic living systems. Adaptation to the seasonal variation in daylength resulted in photoperiodic control of development with a circadian rhythm in energy conservation and transformation to optimise energy harvesting by photosynthesis. Photosynthesis on the other hand acts as a light-dependent metabolic regulator via redox signals in addition to specific photoreceptors like phytochromes and cryptochromes. Finally, redox control integrates rhythmic gene expression in chloroplasts, mitochondria and the nucleus. The circadian rhythmic cell (cyanobacterial and eukaryotic) is a hydro-electro-chemical oscillator synchronised by the daily light – dark cycle with temporal compartmentation of metabolism and a network of metabolic sequences to compensate for oxidative stress in adapting to the light environment e.g. by separating N-fixation from oxygen production. In Chenopodium rubrum L. a circadian rhythm in overall energy transduction has been observed. This rhythm results from an oscillatory network between glycolysis and oxidative phosphorylation coupled to photophosphorylation. This network produces a circadian rhythm in adenylate energy charge and redox state (NADP/NADPH2). The nucleotide ratios themselves could, as rate effectors in compartmental feedback, fulfil the requirements for precise temperature-compensated time keeping. The integration of metabolic activity of Chenopodium plants on a hydraulic-electrochemical level is represented by a diurnal rhythm in compound surface membrane resting potential. Using molecular genetic techniques, research of the last 30 years has come to the conclusion that the core oscillator of circadian systems should reside in transcriptional and translational control loops (TTCL) involved in feedback regulation of clock genes. Considering the evolution of metabolic networks in response to environmental constraints, we proposed (Wagner & Cumming 1970; Wagner et al. 1998) that circadian rhythms in redox state and phosphorylation potential, as an output from the network of energy transduction (Singh 1998), should be gating the TTCL for the circadian rhythmic production of proteins needed in the metabolic networks. A similar concept has been advanced for metabolic control of human circadian rhythms, assuming that the redox state of cells should be the driving effector (Rutter et al. 2002) of the physiological clock.

Rhythmic stem extension growth and leaf movements as markers of plant behaviour: the integral output from endogenous and environmental signals.

With the model systems Chenopodium rubrum (short-day plant) and Chenopodium murale (long-day plant), growth and behaviour have been studied in response to photoand thermoperiod. With time-lapse photography, rhythmic integration of the plant as a whole could be monitored. Upon photoperiodic flower initiation, rhythmic stem extension rate (SER) and leaf movement (LM) change their phase relationship in a specific way. Flower induction correlates to a threshold value for the ratio between integral growth during the dark time span and integral growth during the light time span. This precise output displayed in the growth pattern of the plant is therefore an accurate reflection of all available environmental inputs. Analysis of flower induction in Chenopodium spp. showed that, 2 h after the end of the critical dark period, the patterns of cytoplasmic pH and Ca2+ change at the shoot apical meristem (SAM), possibly indicating the arrival of the flower-inductive signal. Changes in LEAFY and aquaporin expression can also be recorded during this phase. The perception of a flower-inducing dark period probably leads to a change in electrochemical, hydraulic signalling between the leaves and SAM, thereby determining polarity in the whole plant and paving the way for “florigen”, the flower-inducing hormone postulated in 1936 but still undiscovered. A rhythmic integration over the whole plant, as seen for SER and LM, most likely involves modulation of turgor pressure via stretch-activated ion channels and concomitant changes in membrane potential, making the plant a hydro-electrochemical signal transducer. Regulation of hydraulics and electrochemistry, two coupled physicochemical processes, was an achievement of early evolution as well as metabolic circadian regulation of transcriptional S. Mancuso and S. Shabala (Eds.) Rhythms in Plants: Phenomenology, Mechanisms, and Adaptive Significance

Control of Plant Development by Hydro-Electrochemical Signal Transduction: a Means for Understanding Photoperiodic Flower Induction

The hypothesis that flowering involves a specific stimulus is based upon the demonstration that (a) in photoperiodism the flowering response depends upon the day length conditions given to the leaves, whereas the response occurs in the apices, and that (b) a floral stimulus can be transmitted via a graft union from an induced partner (donor) to a non-induced one (receptor). Transmission of the floral stimulus by grafting has been demonstrated within various photoperiodic response types, as well as between different photoperiodic response types in interspecific and intergeneric grafts. The physiological evidence for a floral stimulus is clear-cut, but up to now the nature of the stimulus has remained obscure (Bernier 1988). The specific kind of photoperiodic behavior depends very much on the exact environmental conditions, as was shown for four different North American ecotypes of Chenopodium rubrum (Tsuschiya and Ishiguri 1981). The southern ecotypes display an obligate short-day behavior under white (W), red (R) and blue (B) light. The most northern ecotype is day neutral in B and W and has an amphiphotoperiodic response in R light. Another northern ecotype has an amphiphotoperiodic response in B and a short-day response in W and R light. The amphiphotoperiodic response in B is modified to day neutral by changing the temperature from 20 to 12 °C. These data clearly indicate that photoperiodic behavior is extremely flexible in adapting to specific environmental conditions. Irrespective of the flexibility of plants in modifying their photoperiodic behavior in adapting to specific environmental conditions as just mentioned, the following essentials of the photoperiodic reaction have to be kept in mind as a basis for further considerations:

Rhythmic Stem Extension Growth and Leaf Movements as Markers of Plant Behavior: How Endogenous and Environmental Signals Modulate the Root–Shoot Continuum

With the short-day plant Chenopodium rubrum and the long-day plant Chenopodium murale, growth and behavior have been studied in response to photo- and thermoperiod. With time-lapse photography, rhythmic integration of the plant as a whole could be monitored. Upon photoperiodic flower initiation, rhythmic stem extension rate (SER) and leaf movements (LM) change their phase relationship in a specific way. Flower induction correlates with a threshold value for the ratio between integral growth during the dark-time span and integral growth during the light-time span. This precise output displayed in the growth pattern of the plant is therefore an accurate reflection of all available environmental inputs. Analysis of flower induction in Chenopodium spp. showed that 2 h after the end of the critical dark period, the patterns of cytoplasmic pH and Ca2+ change at the shoot apical meristem (SAM), possibly indicating the arrival of the flower-inducing signal. Changes in LEAFY (a florigenic transcription factor) and aquaporin expression can also be recorded during this phase. The perception of a flower-inducing dark period leads to a change in electrochemical, hydraulic signaling between the leaves and SAM, thereby determining polarity in the whole plant and paving the way for “florigen” . Leaves from flowering plants can be grafted on non-induced plants (short- or long-day species) to induce flowering in the recipient plant. Flowering could even be induced using a different donor and recipient species (inter-species signaling). A rhythmic integration over the whole plant, as seen for SER and LM, most likely involves modulation of turgor pressure via stretch-activated ion channels and concomitant changes in membrane potential, making the plant a hydro-electrochemical signal transducer. Regulation of hydraulics and electrochemistry, two coupled physicochemical processes, was an achievement of early evolution as well as metabolic circadian regulation of transcriptional translational control loops (TTCL). Circadian rhythms (CRs) in energy metabolism are gating inputs and outputs to the TTCL, resulting in a CR of protein synthesis and turnover. Evolution of latitudinal ecotypes with different CR period lengths will depend on specific proteins, as is evident from early crossing experiments. The control of the ionic composition of the cell is crucial for the survival and requires energy to maintain a resting potential of the plasma membrane. This, in turn, enables the generation of action potentials and, hence, a fast systemic communication between plant organs, in particular the root and shoot meristems (RAM and SAM).