Effect of induced changes in membrane permeability on the defence response of Chlorella vulgaris to infection by Acholeplasma laidlawii☆
Introduction
Plant defence is currently an important field for study, not least because of its implications for agriculture [1]. Chlorella is extensively used as a suitable model for investigating this problem [2], [3], [4], [5] because of its structural and physiological similarity of these protists with higher plants. To mimic the defensive response to infection, we have been using a model system in which the microalgal cells of Chlorella vulgaris are challenged with the cells of the mollicute (mycoplasma), Acholeplasma laidlawii [6], [7]. According to these studies, the Chlorella cells reacted to the presence of the mycoplasma by the enhanced production of active oxygen species, which was manifest by simultaneous increases in the rates of heat production to give the so-called heat burst, oxygen uptake in the dark to give the respiratory burst and oxygen evolution as an indicator of the apparent photosynthetic rate in the light. It is important to note that the cascade of stress responses of infected Chlorella cells with the mycoplasma was similar to that found in higher plants [8], [9].
It is obvious that the interface between the pathogen and its host is the cell wall and its subjacent plasma membrane. It is known that the initial signals of the awareness of pathogen infection in plants occur at the interaction between secreted elicitors and trans membrane receptors of both the cell wall and the plasma membrane [10], [11], [12]. The latter plays a crucial role in the reception and transduction of environmental signals, which activates an amplified intracellular signal cascade within the cell leading to a particular response. Among the important non-specific changes of plant cells to pathogen stress are:
- 1.
intensification of biopolymers and lipid catabolism;
- 2.
the change of membrane permeability and as a result the disruption of ion homeostasis;
- 3.
the increase of free radicals content;
- 4.
the alterations of energetic processes rate [10], [11], [12], [13].
For a more complete understanding of the mechanisms of the cellular response to mycoplasma infection, Gd3+, RNase, and ATP were used to modify the ion permeability of plant cells plasma membrane in the following ways. Gd3+ is a specific inhibitor of the mechanosensitive channels in the plasma membrane that does not penetrate into cells [14], [15], [16]. It is thought that the action of RNase action may be due to the degradation of high molecular weight RNA to give lower molecular weight RNA that possesses the enzyme activity to affect embrane permeability [17], [18], [19]. Thus, RNases may take part in defence reactions to pathogen attack [20], [21]. ATP in the living cell carries out not only the energetic role but is important in the formation of such signalling molecules such as cAMP and cGMP as well as phosphorylation cascades, catalyzed by membrane kinases [22], [23], [24].
Alterations in the distribution of ions between the cytoplasm and the external environment may cause changes in the energy metabolism of the cells because ion homeostasis is an active, energy-requiring process. In addition, these alterations may cause the initiation of energy-consuming defence mechanisms, which are tightly related to the intensity of the defence reactions [25], [26], [27]. The metabolic rate of cells and organisms is directly proportional to the instantaneous heat flow rate [28] and thus applying the calorimetric method ensures a complete description of the catabolic changes accompanying the defence against infection.
If oxygen uptake measurements are undertaken simultaneously with direct calorimetry, then it is possible to discriminate between aerobic and anaerobic metabolic processes. Consequently, measurements were taken of the effects of the agents on the oxygen uptake rate. One of the basic mechanisms of non-specific defence of plants on pathogenic attack is the increase in the production of active oxygen species that include the superoxide anion radical [29], [30], [31], [32]. The enhanced generation of the superoxide is likely to have been the most important of the primary defensive responses of the host to the mycoplasma infection. As the viability of the host on infection by the pathogen may be limited by the energetic cost, we studied the rate of photosynthesis. Uniquely, this transforms absorbed light energy into the chemical energy of metabolites and causes the formation of carbohydrates used by organisms as the energy source for catabolic and anabolic processes, with the former providing the energy for the latter.
The aim of this research is to study key energetic defence processes of Chlorella cells infected with mycoplasma by the use of specific membrane permeability modulators.
Section snippets
Experimental
The unicellular microalga Chlorella vulgaris and the Mollicute Acholeplasma laidlawii were the objects for this investigation. Chlorella cells were grown in Tamiya medium, pH 6.8–7.2 [33] at 30 °C, and illuminated at 1 × 104 lx with a photoperiod of light/dark of 12/12 h. Cell suspensions were bubbled with 0.3% CO2 in air. The optical density was maintained at (1–1.5 × 108 cells/ml).
A. laidlawii was grown at 37 °C in test tubes using the Edward medium [34]. Before each experiment, the mycoplasma
Results
As can be seen in Fig. 1, as soon as the vessels were equilibrated to 37 °C it could be seen that the instantaneous rate of heat production of the Chlorella cells treated with mycoplasma was 250% greater than in the control. The rate of heat production of algal cells treated with Gd3+ was 15–20% lower than the control 40 min after addition of Ga3+ to the Chlorella suspension and this difference continued throughout experiment period. The amount of heat production by Chlorella suspension treated
Discussion
As a general feature, photosynthesising organisms react to the action of various pathogens as well as other extreme factors with a cascade of stress responses. The signals of the awareness of the pathogen infection by them are universal. The interaction between elicitors, exocytosed in medium and transmembrane protein receptors of plasma membranes of the host play the role of the initial reaction. The signals trigger the chain of the processes of induction and regulation of phytoimmunity [10],
Conclusions
Summarizing the data, it can be concluded that the defense reactions of microalgal cells to infection require additional energy. It is proposed that the conditions decreasing the energetic status of plant cells reduce the phytoimmunity of the organisms to the infection. So, Gd3+ ions rapidly decreased the heat production rate of the infected Chlorella. RNase and ATP, on the other hand, increase respiration and photosynthesis, as seen by the increase in the rate of heat production, and thus
Acknowledgements
The investigation was carried out with the financial support of INTAS project, 99–01390 (Project Co-ordinator, Dr R.B. Kemp).
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Presented at the thirteenth meeting of the International Society for Biological Calorimetry, Wurzburg-Veitschochheim, Germany, 27 September to 1 October 2004.