Departamento de Biología, Facultad de Ciencias, Universidad Autónoma de Madrid, Madrid 28049, Spain
Correspondence
F. Fernández-Piñas
francisca.pina{at}uam.es
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ABSTRACT |
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INTRODUCTION |
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Cyanobacteria are a large group of photosynthetic, oxygen-evolving prokaryotes. Many of them are also capable of fixing atmospheric N2, a process that requires nitrogenase to be protected from oxygen. The filamentous cyanobacterium Anabaena sp. PCC7120 differentiates specialized cells called heterocysts that create a microoxic environment for nitrogen fixation; heterocysts normally form at semi-regular intervals along the filaments, following a developmental pattern, as a response to nitrogen deprivation. Heterocyst differentiation follows a specific scheme and requires global changes in gene expression (reviewed by Wolk et al., 1994; Wolk, 1996
, 2000
, Golden & Yoon, 1998
, 2003
) but an overall model of the regulatory networks controlling development remains elusive. In the regulation of early events in the process of differentiation, the expression patterns of several genes have been studied and a few have been placed into an ordered sequence; among these, hetR is a key regulator of heterocyst development that starts to be activated within 2 h and is already active in spaced cells in the filament within 3·5 h of nitrogen deprivation. Anabaena sp. PCC7120 hetR mutants fail to differentiate (Wolk et al., 1994
, Black et al., 1993
). hetR encodes an unusual serine-type protease that has been suggested to have a calcium-modulated activity (Zhou et al., 1998
; Dong et al., 2000
).
The involvement of a second messenger in the process of heterocyst development is not clear. Hood et al. (1979) reported that in Anabaena variabilis the cAMP levels increased fourfold after nitrogen removal but more recent studies have cast doubt on the role of cAMP in nitrogen metabolism in cyanobacteria (Cann, 2003
). However, growing evidence indicates that calcium might truly be involved in heterocyst development; in early studies Smith et al. (1987)
noted that the external concentration of Ca2+ and the presence of several calcium agonists influenced heterocyst frequency in Nostoc PCC 6720. Later studies found a correlation between heterocyst frequency and different treatments that affect the accumulation of Ca2+ in cells during the process of differentiation (Smith, 1988
; Smith & Wilkins, 1988
; Zhao et al., 1991
; Onek & Smith, 1992
), hence reinforcing the hypothesis that Ca2+ might have a regulatory role in the differentiation of heterocysts.
We have therefore investigated intracellular free calcium changes following nitrogen deprivation in strain Anabaena sp. PCC7120(pBG2001a), which constitutively expresses recombinant aequorin (Torrecilla et al., 2000). We have found a Ca2+ transient with a specific calcium signature (spatial and temporal characteristics of stimuli-specific calcium transients) that starts shortly after nitrogen deprivation. We have also shown that a hetR mutant retains the calcium transient. Finally, evidence is presented that indicates a correlation between the suppression or alteration of such calcium signals and a subsequent early arrest of heterocyst differentiation.
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METHODS |
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In vivo aequorin reconstitution and luminescence measurements.
For aequorin luminescence measurements, in vivo reconstitution of aequorin was performed by the addition of 2·5 µM coelenterazine (Molecular Probes) to cell suspensions (15 µg chlorophyll ml1) and incubation for 4 h in darkness and with shaking, as previously described (Torrecilla et al., 2000). Excess coelenterazine was removed before Ca2+ measurements were taken.
Luminescence measurements were made using a digital luminometer with a photomultiplier (BioOrbit 1250). Reconstituted cell suspensions (0·5 ml) in a transparent polystyrene cuvette were placed in the luminometer and luminescence was recorded every 1 s for the duration of the experiment.
Calibration of the [Ca2+]i changes requires the knowledge of the total available amount of reconstituted aequorin in cell suspensions (Lmax) at any one point in time during the experiment, as well as the running luminescence (L0). For estimation of total aequorin luminescence, the remaining aequorin was discharged at the end of the experiment by the addition of 0·5 ml 100 mM CaCl2 and 5 % (v/v) Triton X-100. Rate constants of luminiscence (L0 Lmax1) were determined for each point along the experiment, and [Ca2+]i was calculated by using calibration curves obtained for aequorin extracted from the recombinant strain of Anabaena sp. PCC7120, according to Torrecilla et al. (2000).
Nitrogen deprivation experiments.
Before nitrogen deprivation, cell suspensions of Anabaena sp. PCC7120(pBG2001a) grown with nitrate or ammonium were reconstituted with coelenterazine. To induce heterocyst formation, reconstituted cell suspensions were washed four times with combined-nitrogen-free medium (medium BG110, buffered with 25 mM HEPES/NaOH to pH 7·20), followed by incubation in the same combined-nitrogen-free medium. When the experiments required total absence or a lower or higher concentration of Ca2+ than the standard, a modified BG110 with the required calcium concentration was used for nitrogen deprivation. When needed, 200 µM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA; Sigma), 5 µM compound A23187 (Sigma) or 5 µM trifluoperazine (TFP; Sigma) was added at this step. To facilitate cell loading of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy-methyl ester) (BAPTA-AM; Molecular Probes), it was added at a final concentration of 300 µM 1 h before aequorin reconstitution and then again at this step.
Bright-field and fluorescence micrographs were taken with an Olympus BH-2 microscope equipped with epifluorescence and a digital camera Leica DC 300F. Proheterocysts and mature heterocysts were clearly discernible at 10 and 20 h, respectively, after nitrogen deprivation. Heterocyst frequency was calculated as described by Smith & Wilkins (1988).
Statistical procedures.
All tests of statistically significant differences between datasets were performed using Student's t-tests or analysis of variance at P<0·05 with the program SigmaStat. All data were obtained from a minimum of three repetitions for each assay situation.
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RESULTS |
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To check whether this calcium transient was a specific response to nitrate deprivation or a response to a lack of combined nitrogen, cultures were grown for several days in a modified BG11 medium containing 5 mM ammonium (NH4Cl) instead of nitrate as the nitrogen source. The subsequent removal of ammonium from the medium gave rise to a calcium transient (Fig. 1a) with very similar features to that described above for removal of nitrate, though the maximum [Ca2+]i reached was slightly lower, 0·31±0·11 µM (n=5). Likewise, as a control, cell suspensions washed several times with BG11 with ammonium and finally resuspended in the ammonium-containing medium did not show any change in the levels of [Ca2+]i (data not shown). Also, a shift in the medium from nitrate to ammonium did not induce the calcium transient (not shown).
The experimental procedures for these measurements, which rely on the continuous detection of the luminescence from the cell suspension, meant that the sample had to be kept in total darkness for several hours. To test the possible effect of such absence of light during the time of measurement on the course of heterocyst differentiation, cell suspensions were transferred after measurements from the luminometer chamber to a thermostatic chamber at 28 °C under white light at a light intensity of 65 µE m2 s1. After 20 h, it was verified that the morphology and the frequency of the heterocysts that had differentiated was not significantly different from those of the sample that had been kept under continuous light (frequencies of 10·2 % and 11·0 %, respectively). This result indicated that exposure to darkness for 45 h after nitrogen withdrawal did not affect the process of differentiation. Nonetheless, since light is needed for heterocyst differentiation to take place correctly in the obligate autotroph Anabaena sp. PCC7120 (Wolk et al., 1994), we checked whether the observed calcium signature also appeared under light conditions. For this purpose, discontinuous recording of luminescence data at specific intervals was performed. Thus, a cell culture deprived of combined nitrogen was divided into several aliquots and kept under light at a light intensity of 65 µEs m2 s1. At specific time intervals, aliquots were transferred, one by one, to the luminometer chamber and the stable level of [Ca2+]i was determined. As can be seen in Fig. 1(b), a
defined calcium transient also occurred under light conditions, with a magnitude and duration very similar to that described when samples were kept in the luminometer continuously for several hours after nitrogen deprivation. The maximum stable calcium level reached for the assayed times was 0·45±0·11 µM (n=5), which corresponded to a time of 2 h 35 min. Thus, the observed calcium transient seems to be a physiological response to combined-nitrogen deprivation.
The data presented above strongly suggest that the observed calcium transient is triggered by the withdrawal of the source of combined nitrogen from the external medium. Nevertheless, with the aim of further corroborating the specificity of the calcium response, we investigated the possibility that deprivations of other nutrients, such as iron or phosphate, might also induce an analogous early calcium transient. Reconstituted cultures of Anabaena were washed several times with modified BG11 prepared with the omission of the corresponding salts (ferric-ammonium citrate or K2HPO4) and finally resuspended in the medium without iron or phosphate. In both cases, the [Ca2+]i of the cell suspensions remained at basal level (not shown). Nonetheless, although the [Ca2+]i was monitored for the subsequent 67 h after the deprivation of such nutrients, the possibility of the occurrence of calcium transients later on, once the iron or phosphate deficiencies are more pronounced, cannot be rejected.
Determination of the possible cellular origin of the calcium signature induced by combined-nitrogen deprivation
In order to establish the source of calcium responsible for the observed [Ca2+]i transient, nitrogen deprivation experiments were performed in which cells were finally resuspended in BG110 medium either without calcium and with 200 µM of the extracellular calcium chelator EGTA or supplemented with calcium up to 5 mM. As shown in Fig. 2, extracellular calcium depletion was not reflected by a diminution in or suppression of the observed calcium transient; the transient had very similar features, in terms of amplitude and kinetics, to those seen with the standard calcium concentration (the highest free-calcium concentration reached was 0·32±0·10 µM [n=5]). Likewise, the increase in the external calcium concentration up to 5 mM had no significant effect on the magnitude and kinetics of the calcium transient. Therefore, these data suggest that the appearance of the calcium transient is independent of an extracellular source of calcium and thus, the main source of calcium seems to be intracellular.
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Two sets of contrasting experiments were performed with the Ca2+ agents: firstly, agents were added just after nitrogen deprivation so that the agent was present from the beginning of the differentiation process and should, therefore, alter the calcium transient; and secondly, agents were added once the calcium transient was over and the basal intracellular calcium level had recovered (approximately 6 h after nitrogen removal). For both sets of experiments, changes in intracellular calcium levels were continuously measured in the luminometer and, in parallel, the course of differentiation was monitored by bright-field and fluorescence microscopy. Fluorescence microscopy was used to check for patterned loss of red fluorescence which occurs due to the degradation of phycobiliprotein at an early stage of development in cells committed to become heterocysts (Wolk, 2000; Wood & Haselkorn, 1980
). As a control, both Ca2+ monitoring and microscopy observations were performed in cell suspensions not treated with the calcium agents. Also, the three agents at the tested concentrations did not have any significant effect on vegetative growth in nitrate-containing medium during the experimentation time (not shown).
Effect of the calcium ionophore compound A23187.
Previous results in our laboratory have shown that compound A23187 causes a significant elevation of the Ca2+ transients induced by environmental shocks in Anabaena sp. PCC7120 (Torrecilla et al., 2001). As represented in Fig. 3
, when the cell cultures were pretreated with 5 µM A23187 just after nitrogen deprivation, the shape of the induced calcium signature changed dramatically, resulting in a significant rise in the magnitude of the Ca2+ elevation (0·74±0·26 µM [n=6] versus 0·39±0·12 µM [n=32] in the control) and in a longer duration of the transient, as 5 µM of the ionophore did not restore [Ca2+]i basal levels even after 5 h (Fig. 3a
). The effect of the addition of 5 µM A23187 immediately after nitrogen removal on heterocyst differentiation was remarkable; no heterocysts or proheterocysts were observed 24 h after nitrogen removal, or even 3 days after nitrogen removal, and no loss of phycobiliprotein fluorescence was observed either (Fig. 3b, c
), indicating that heterocyst differentiation had been arrested at an early stage of development. Control cultures without A23187 showed a normal pattern of heterocyst differentiation (Fig. 3d, e
). However, when the ionophore was added after the Ca2+ transient was over and [Ca2+]i basal levels had been restored, it did not modify calcium basal levels except for a short spike that represented a small, mechanically induced Ca2+ increase upon ionophore injection (Torrecilla et al., 2000
) and did not have any effect on the process of heterocyst differentiation (Fig. 3f
h). The data obtained with the calcium ionophore A23187 indicated that a significant alteration of the specific calcium signature, by means of a change in signal amplitude and duration, is correlated with an early inhibition of heterocyst differentiation. In addition, these results rule out the possibility that the ionophore itself (Fig. 3g, h
) could impair differentiation.
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Calcium signals in a hetR mutant of Anabaena sp. PCC7120
A recombinant strain of an Anabaena sp. PCC7120 hetR mutant expressing apoaequorin was constructed. Due to the critical and early involvement of hetR in heterocyst differentiation, we wanted to know whether the observed calcium transient following nitrogen deprivation was induced in the hetR mutant. As Fig. 6 shows, a calcium transient with quite similar features regarding amplitude and kinetics to those found in the wild-type strain could be detected, suggesting that the calcium transient may be an upstream event to hetR expression, being involved in an earlier step of the differentiation process.
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DISCUSSION |
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We have observed that upon nitrogen deprivation, resulting from either removing nitrate or ammonium, an early calcium transient is triggered that constitutes a specific calcium signature that differs from the calcium signatures observed upon exposure of the cyanobacteria to other environmental stresses such as heat and cold shock, acid shock, osmotic and salt stress and light-to-dark transitions (Torrecilla et al., 2000, 2001
, 2004
). Compared with the previously tested environmental stimuli, the observed monophasic and bell-shaped Ca2+ signal reaches a modest magnitude (0·39 µM versus 3·10 µM after cold shock or 2·30 µM after a hyperosmotic shock) but lasts nearly 4 h (the transient duration of other environmental stimuli ranges from 34 min to over 1 h). Another main difference is that the calcium source responsible for the calcium elevation induced after nitrogen deprivation seems to be entirely intracellular, while for most of the other tested stimuli the calcium source appears to be mainly or entirely extracellular.
Although it has been proposed that calcium may merely act as a chemical switch in signal transduction (Scrase-Field & Knight, 2003), it is generally assumed that the spatio-temporal components of the increase in [Ca2+]i or Ca2+ signature dictates the outcome of the cellular end response (Ng & McAinsh, 2003). There are a few examples in the literature where modulation or alteration of specific calcium signatures in eukaryotic cells correlates with a concomitant modulation of physiological responses (Dolmetsch et al., 1997, 1998
; Allen & Schroeder, 2001
; Gu & Spitzer, 1995
; Allen et al., 2000
, 2001
). We have also manipulated the Ca2+ signal recorded upon nitrogen deprivation by using Ca2+ agents that affect calcium homeostasis (the ionophore A23187, the calmodulin inhibitor TFP and the intracellular calcium chelator BAPTA-AM) and have monitored the subsequent effect on the process of differentiation. We have found that suppression, magnification or poor regulation of the signal is correlated with an inhibition of heterocyst differentiation. However, addition of the calcium agents once the signal is over does not have any significant effect on Ca2+ basal levels, and heterocyst formation is not affected.
Zhao et al. (1991) also reported that the ionophore A23187 in the presence of external calcium inhibited heterocyst differentiation. However, if they added the ionophore 67 h after nitrogen deprivation, a time that noticeably matches with the end of the calcium signal that we report here, nitrogen differentiation was not arrested. They did not monitor intracellular free-calcium levels, nor could they detect the specific calcium signature. Subsequently they concluded that calcium may have a role in suppressing an early stage of heterocyst differentiation. However, our results suggest that a specific calcium signal with a defined set of temporal and kinetic parameters may be required for the process of heterocyst differentiation.
The observed effect of the eukaryotic calmodulin inhibitor TFP on the regulation of the Ca2+ signal and on heterocyst differentiation suggests the presence of calmodulin or calmodulin-like activities in Anabaena sp. PCC7120. Since 1984 there has been immunological as well as biochemical evidence for the presence of cyanobacterial calmodulins (Kerson et al., 1984); Pettersson & Bergman (1989)
previously found a 17 kDa polypeptide with calmodulin-like activity in Anabaena sp. PCC7120 and in Anabaena cylindrica. Recent genomic analyses also revealed the existence of cyanobacterial proteins with Ca2+-binding domains (Michiels et al., 2002
; http://smart.embl-heidelberg.de).
The fact that a hetR mutant of Anabaena sp. PCC7120 also displays the calcium signature upon nitrogen deprivation suggests that the Ca2+ signal may be an upstream event that occurs before the induction of the expression of hetR, which is already active in spaced cells within 3·5 h of nitrogen deprivation. During the early phases of differentiation, intracellular protein degradation increases several-fold (Wood & Haselkorn, 1980); proteolysis appears to supply amino acids for heterocyst-specific protein synthesis and must play an important role in the differentiation process. Two proteolytic activities have been related to this process: a Ca2+-stimulated protease degrading numerous proteins of vegetative cells in vitro, and a protease apparently specific for phycobiliproteins (Wood & Haselkorn, 1980
).
Under our experimental conditions, alteration of the observed Ca2+ signal correlated with an early arrest of heterocyst differentiation as evidenced by the absence of patterned loss of phycobiliprotein fluorescence. Therefore it is tempting to correlate the calcium transient with the signalling for proteolysis of phycobiliproteins. In fact, in B. subtilis a Ca2+ influx has been shown to be essential for the early proteolysis that takes place during sporulation (O'Hara & Hageman, 1990; Shyu & Foegeding, 1991
; Dominguez et al., 1999
). However, the relevance of the calcium-dependent protease in the process of heterocyst differentiation remains unclear since Lockau et al. (1988)
and Maldener et al. (1991)
demonstrated that this protease is not essential for the development of functional heterocysts. Also, the non-differentiating hetR mutant of Anabaena sp. PCC7120 shows no loss of phycobiliprotein fluorescence but retains the Ca2+ signal, implying that the observed signal may not be directly related to phycobiliprotein degradation.
As a final point, it should be noted that the calcium transients presented in this work reflect the luminescence emerging from the whole population of filaments in the cell suspension, which raises two key questions to be taken into account. Firstly, the data can only be interpreted in terms of the mean [Ca2+]i in the totality of cells in the sample, with no indication of the distribution of Ca2+ along the filaments. The method used to detect aequorin luminescence does not provide information on whether the increase in [Ca2+]i is confined to a fraction of cells within the filament or whether the whole filament contributes evenly to the signal. In fact, in order to detect which cells in the filament were undergoing the increase in intracellular calcium, we tried to image, at the single cell level, calcium dynamics in our recombinant Anabaena strain, but we failed, probably due to the low quantum yield of aequorin luminescence. At present, we are trying to express in Anabaena a GFP cameleon construct (yellow cameleon 2.1) that has allowed successful calcium single-cell imaging in plant cells (Allen et al., 1999). Secondly, the observed calcium transient may be covering more complex individual dynamics such as calcium oscillations, which would be hidden by cells that are not in phase with each other. Also, further research is needed to identify the initial sensor or mechanism that triggers the Ca2+ signal upon nitrogen deprivation as well as the downstream responses, in order to better define the role of calcium in heterocyst differentiation.
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ACKNOWLEDGEMENTS |
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Received 15 June 2004;
revised 22 July 2004;
accepted 3 August 2004.
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