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Fate of a single cell in a developing plant meristem is determined by positional information. Coordination of responses of tissues to environmental signals depends on intercellular communication. Stable calcium gradients, or ´ultraslow´ waves, were observed in different organisms and seem to be a general feature in developmental coordination of cell populations and tissues, e.g. in zebrafish embryos, developmental pattern formation is preceded by calcium gradients. Signal transduction in cells, tissues and organs is not linear, but involves a dynamic network of interactions. Therefore, understanding the interplay of different signalling pathways is of basic interest. There is some literature evidence on possible relations between Ca2+- and NO-signalling and mitochondrial activity and cytoplasmic Ca2+- and pH-regulation.
Visualization of signalling pathways during developmental control is feasible by using in vivo fluorescent probes. Increasing numbers of very specific fluorescent probes which can pass the plasma membrane are not easy to use in plant tissue because of cell walls and the cuticula. We could adapt a series of fluorescent probes for monitoring of plant apical meristems, through variations in loading protocols as well as cutting the meristems in half with a special microtome ´vibratome´. AM-esters of Fluo-3 and Fura Red, and of carboxy SNARF-1 were used for estimation of calcium concentrations and pH, respectively. Mitochondrial activity was visualized using JC-1. In all these cases, simultaneous double-channel detection with CLSM and rationing was used to avoid artefacts from different loading of the dye into the cells. For NO-detection DAF-FM diacetate was used (dyes were from Molecular Probes Europe, Leiden, Netherlands). For more details about the methods and about the fluorescent indicators see: www.probes.com
The significance of specific distribution patterns of calcium concentrations, pH, mitochondrial activity and NO in the apical meristem of Chenopodium rubrum (Fig. 1A-E, J-K) seems very promising for further investigations. The patterns changed after the switch from vegetative development to flowering due to photoperiodic induction (Fig. 1G-I). The change of patterning is visible immediately after the end of an inductive photoperiod and persists for several hours. Differences were quantified using the linescan method (Fig. 1M, N). pH-patterns in vegetative and induced plants were differentially influenced by glucose feeding. Preliminary results on the analysis of the meristem surface using cryo REM (Fig. 1L) are indicative of local turgor differences between cells which might be related to the observed metabolic patterning. Morphogenetic changes during flower induction seem to be preceded by changed calcium patterns, as described in animal embryonegesis, playing together with local changes of turgor and pH, factors known to be involved in plant development.
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Fig. 1: Maps of apical meristems of Chenopodium rubrum, obtained using fluorescent probes and cryo REM. A-E - vegetative plants, half sections; A - calcium (Fluo-3 + Fura Red), B - pH (carboxy SNARF-1), C - mitochondrial activity (JC-1), D - NO (DAF-FM), E - NO after application of elicitor (DAF-FM, SNP). F - calibration scale for [Ca2+] (left) and pH (right); the same scale was used to indicate mitochondrial activity (high~blue, low~red) and NO-concentration (high~red, low~blue). G-I - plants after flower induction, half sections (fluorescent probes as A-C); G - calcium, H - pH, I - mitochondrial activity. J-L - vegetative plants, surface; J - NO (DAF-FM), K - NO after application of elicitor (DAF-FM, SNP), L - cryo REM. N, M - linescan graphs of A and G, resp., quantifying differences of calcium patterning. On behalf of the linescan graph, so called inhomogenenity factor can be calculated, which characterises the pattern. All pattern maps can be similarly quantified.
Signal transduction in photoperiodic flower induction involves the perception of the signal (inductive photoperiod) by the leaves and transport to the target tissue, apical meristem, where morphological changes follow. The earliest events are cell activation in the central zone and enhanced initiation and growth of primordia. pH and calcium play a role in signal transduction as second messengers.
Both pH and [Ca2+] patterns at the apical meristem changes after inductive photoperiod in a short day plant Chenopodium rubrum and in a long day plant Chenopodium murale. Patterning is controlled by phytochrome in both cases, as shown with experiments using red and far red light. As pH patterns change already during the inductive photoperiod, the long-distance signal transduction from the leaves to the apex is very fast. Therefore, the involvement of electrical signals is supposed. Changes are related to the changed morphogenesis on the apical meristem after flower induction: a shift to a more basic pH at the sites of the subsequent initiation of new primordia was observed.
Typical pH and calcium patterns at the apical meristem of Chenopodium rubrum and Chenopodium murale (Fig. 2 and Fig. 3):
- Fine alternating strips (rows of cells with different pH or [Ca2+] (Fig. 1A, B).
- Homogeneous pattern (high average pH and [Ca2+] (Fig. 1G, H).
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Fig. 2: [Ca2+]-patterns in Chenopodium spp.
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Fig. 3: pH-patterns in Chenopodium spp.
Further we are interested in metabolic changes during flower induction: the involvement of glycolysis and respiration. For the studies on respiration we use an another fluorescent indicator, JC-1, which is loaded in the membranes of mitochondria and change its fluorescence depending on the activity of mitochondria (Figs. 1C, I; 4).
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| a) active mitochondria | b) not active mitochondria |
Fig. 4: Activity of mitochondria in the cells of apical meristem of Chenopodium rubrum, visualised using JC-1. On the both images the same cells were scanned in two different wavelengths: > 610 nm (a) und 520-540 nm (b), in which active and not active mitochondria, respectively, are visible. You can see 3 cells on the images, two with high respiration activity (strong fluorescence at the longer wavelength (a) and low fluorescence intensity at the shorter wavelength (b). The cell with not active mitochondria can be well localized at the shorter wavelength (b).
Recently, we have started to study NO-signalling (using a fluorescent indicator DAF-FM - Fig. 1D, E, J, K) and turgor regulation on the apical meristem (using the method of cryo REM - Fig. 1L; in co-operation with Central Laboratory of Microscopy, University of Basel). The aim is to understand the interplay of different signalling pathways involved in the changes at the apical meristem during and after photoperiodic flower induction.
List of Publications
© Dr. J. Normann