Jolana T. P. Albrechtová

Plasma Membrane H + -ATPase Is Involved in Auxin-Mediated Cell Elongation during Wheat Embryo Development

Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H+-ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two–three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H+-ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H+-ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H+-ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.

Hydro-Electrochemical Integration of the Higher Plant — Basis for Electrogenic Flower Induction

The integration of activity of Chenopodium plants on a hydraulic-electrochemical level is expressed by a diurnal rhythm in the resting membrane potential measured with contact electrodes. The membrane state could be gated by the energy state of cells. From earlier studies we compiled evidence in favour of a circadian rhythm in overall energy transduction producing a circadian rhythm in energy charge and redox state (NADPH2/NADP). The ratio of metabolic coupling nucleotides would be relatively temperature independent and thus could fulfil the requirements for precise temperature-compensated time-keeping. The phytochrome photoreceptors, involved in photoperiodic control of development, could via changes in pyridine nucleotide pool sizes and changes in nucleotide ratios regulate transcription-translational loops by redox and phosphorylation controlled transcription factors. Spontaneous action potentials (APs) have been shown to correlate with turgor-controlled growth movements. The accumulation of spontaneous APs at specific times during daily light-dark spans were recorded, giving specific electrophysiograms, representative for flower-inducing and vegetative conditions. It is anticipated that hydraulic changes at the apex leading to flower initiation are mediated by a specific hydro-electrochemical communication between leaves, the shoot apex and the root system. These results have been used to substitute a flower-inducing photoperiod by specific timing of electric stimulation via surface electrodes.

pH-patterning at the shoot apical meristem as related to time of day during different light treatments

Cytoplasmic pH is an important regulatory factor in metabolism. It changes during the cell cycle and during development. The timing of changes in pH-distribution at the apical meristem during three different photoperiods was examined in the short-day plant Chenopodium rubrum in order to study the role of pH in changes of sensitivity of the shoot apex to a photoperiodic flower inducing treatment. pH-changes were visualized in vivo by means of a fluorescent cytoplasmic pH-probe, carboxy SNARF-1 and by confocal laser scanning microscopy. Typical stripy, patchy and uniform patterns could be determined on the basis of pH-patterning at the apical meristem under different photoperiodic treatments. Under continuous light no change in pH-patterning could be observed. The pattern of pH-distribution at the apical meristem specifically changed in response to light-off. During the subsequent dark span pH-pattern changed again after exceeding the critical flower inducing dark period. Responsiveness of pH-distribution at the apical meristem to conditions inductive for flowering is suggestive of the involvement of intracellular pH-changes in the pathway of photoperiodic signal transduction.

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

Tree maturation characterized by in vivo studies in Pinus radiata on pH patterns at the apex and systemic electrophysiological responses.

Pinus radiata plants of different ages (juvenile and adult) were analysed to identify developmental stage specific physiological markers. Patterns of cytoplasmic pH measured in the apical meristem using the fluorescent probe carboxy SNARF-1 and confocal laser-scanning microscopy became more homogeneous during development with the average pH shifting during maturation to more acidic values in all tested genotypes. Electrophysiological responses to external signals were measured as surface compound potential changes using surface electrodes. The responsiveness to light and temperature signals decreased during development in all genotypes, with the exception of a non-flowering one.

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:

Biosystems Analysis of Plant Development Concerning Photoperiodic Flower Induction by Hydro-Electrochemical Signal Transduction

In contrast to the classical hypothesis of photoperiodic flower induction involving a flower-inducing hormone ‘florigen’, it is the goal of this contribution to present the higher plant as a hydraulic-electrochemical signal transducer integrating the plant organs via action potentials in communication with intrinsic and environmental constraints and to replace ‘florigen’ by a frequency-coded hydro-electrochemical signal pattern. Observation of whole plant behaviour by time lapse photography clearly shows rhythmic integration of the main shoot axis and side branches in rhythmic growth as well as in leaf movements. This was observed with short-day ecotypes of Chenopodium rubrum L. and a long-day ecotype of Chenopodium murale L. Upon flower induction the phase relationship between rhythmic SER and leaf movements is altered in a very specific way both in the short-day and long-day plant (Wagner et al., Flowering Newslett, 26:62–74, 1998). The experimental set-up for running these investigations is shown in Fig. 11.1. The recording of surface sum action potentials from the surface of Chenopodium plants is achieved with bipolar electrodes and differential amplifiers similar to the equipment used for recording EEGs and ECGs in medicine. Finally, electrophysiograms (EPGs) can be obtained from various phases of plant development (i.e. juvenility, maturity) to be used for applied purposes like plant characterisation and manipulation in nurseries or glasshouse crops. Rhythmic integration of the whole plant possibly involves modulation of turgor pressure via stretch-activated ion channels and concomitant changes in membrane potential. The perception of a flower inducing dark period might lead to a change in electrochemical signalling between leaves and the shoot apical meristem (SAM) and thus represent ‘florigen’. The switch from the vegetative to the flowering state is a threshold response, systemic in nature and involving not only the apical meristem but also the axillary buds. Thus we believe that the flower inducing signal may be electrical in nature, requiring a holistic biosystem analysis for quantitative ilucidation.

Use of Fluorescent Probes and CLSM for pH-Monitoring in the Whole Plant Tissue. pH-Changes in the Shoot Apex of Chenopodium rubrum Related to Organogenesis

Cytoplasmic pH changes were reported to be related to many life processes (Felle 1989). In many organisms pH changes specifically during the cell cycle and during the life time: activation and juvenility being associated with pH-increasing, and quiescent state and senescence with pH-decreasing (Felle 1989, Ross 1992). Data obtained in animal cells point to an important role of the cytoplasmic pH in regulation of metabolic processes (Busa and Nuccitelli 1984, Nucitelli and Deamer 1982) and in the activation of enzymes playing a role in signal transduction cascades (Felle 1989). There are not many studies carried out on plant material; however, all of them suggest the importance of intracellular pH in regulation of cell cycle and development (Felle and Berti 1986, Gendraud and Lafleuriel 1983, Roos and Slavik 1987, Tort and Gendraud 1984). Local pH-changes are related to changes of membrane potential and proton fluxes (Blatt and Slayman 1987, Felle 1987, Felle and Berti 1986, Frachisse et al. 1988), which lead to a hypothesis of pH playing a role as a second messenger in signal transduction between plasma membrane and cytoplasm (Felle 1989).