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CHAPTER SEVEN

Oxygen Evolution and Chlorophyll Fluorescence Under Extreme Desiccation in the Aquatic Bryophyte Fontinalis antipyretica Ricardo Duarte Cruz1, Cristina Branquinho2,3, and Jorge Marques da Silva1

Abstract Our objective was to measure the impact of different levels and periods of desiccation in photosynthesis and respiration in the aquatic bryophyte Fontinalis antipyretica, using oxygen evolution, chlorophyll a fluorescence and ion leakage techniques. We found a substantial increase in O2 consumption during the dark that was not inhibited by the mitochondrial inhibitors myxothiazol and propyl gallate. Photosynthetic activity decreased severely under extreme desiccation as shown by oxygen evolution and chlorophyll fluorescence parameters. F. antipyretica showed to be extremely sensitive to the imposed desiccation conditions being unable to recover its normal metabolic activity. This can be the result of cellular membrane damage since a substantial electrolyte leakage was observed.

1

Universidade de Lisboa, Faculdade de Ciências and Centro de Engenharia Biológica, Campo Grande, Edifício C2, Piso 4. 1749-016 Lisboa, Portugal 2 Universidade de Lisboa, Faculdade de Ciências, Centro de Ecologia e Biologia Vegetal (CEBV), Campo Grande, Edifício C2, Piso 4, 1749-016 Lisboa, Portugal 3 Universidade Atlântica, Antiga Fábrica da Pólvora de Barcarena, 2730-036 Barcarena, Portugal J.F. Allen, E. Gantt, J.H. Golbeck, and B. Osmond (eds.), Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 1425–1430. © 2008 Springer.

Keywords Mosses, oxidative burst, photosynthesis, electrolyte leakage, respiration

Introduction In face of the current climate changes, drought has been increasing worldwide. In particular it is expected that the Mediterranean region suffers in future with more severe and prolonged droughts. This will be particularly important in intermittent Mediterranean streams. Fontinalis antipyretica is an aquatic bryophyte of river springs which is adapted to intermittent streams being able to loose or gain water in response to environmental changes in particular during the dry season (Glime and Vitt 1984). During the drying process, terrestrial bryophytes suffer several impacts including chloroplast ultrastructural changes, mitochondrial deformation, vacuolar breakdown into smaller vesicles (Glime 2007) and membrane damage which compromises cell structure and induces ion leakage (Brown and Buck 1979; Oliver et al. 2005). The impacts may last only for a few minutes or may cause permanent damage (Oliver and Bewley 1984). During rehydration loss of nutrients may occur

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Oxygen Evolution and Chlorophyll Fluorescence Under Extreme Desiccation

(Gupta 1977). Terrestrial desiccation-tolerant bryophytes protect their cell membranes from oxidative destruction. In these species, there is induction of H2O2 production in light, reduction of the loss of K+, and reduction in oxygen release from photosystem II (Glime 2007). On the other hand, dehydration of desiccation-intolerant bryophytes causes disruption of cellular membranes, leakage of cytoplasmic solutes and protein denaturation due to the accumulation of reactive oxygen species (Oldenhof et al. 2006) and the removal of water from biomolecules (Crowe et al. 1992). The rate of desiccation is important since even desiccation-tolerant plants require a slow drying process to induce protective mechanisms that allow tissues to survive dehydration (Glime 2007). Most of the information concerning the mechanisms of tolerance to desiccation was obtained in terrestrial bryophytes. Our objective was to measure the impact of different levels and periods of desiccation in photosynthesis and respiration of the aquatic bryophyte Fontinalis antipyretica, using oxygen evolution, chlorophyll a fluorescence and ion leakage techniques.

Materials and methods Plant material. Samples of the aquatic moss Fontinalis antipyretica L. ex Hedw. were collected at Serra de S. Mamede, Portugal, and brought to the laboratory under cooling conditions. Mosses were rinsed in distilled water and grown in a growth chamber at 17°C day/12°C night, 20–30 µmol m−2 s−1 PAR and photoperiod of 16 h. Ten shoot moss tips with 1 cm each (three replicates) were collected for each measurement. Desiccation conditions. During the desiccation period, samples were maintained under controlled temperature (21°C) and at different relative humidities (RH) (23%, 50%, 95%), using saturated salt solutions of KC2H3O2, Ca(NO3)2.4H2O and K2SO4, respectively, for several periods of time (30 min up to 40 days). After desiccation, recovery was made through immersion in 1 mL deionised water with 20 µL 5 mM KHCO3 at 17°C, during oxygen evolution measurements.

Oxygen exchange measurements. Measurements of oxygen exchange were done before and after desiccation induction using a Clark type liquid phase oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK), for 10 min in the dark, 10 min under PAR 46 µmol m−2 s−1 and in the dark again for 5 min. Gross photosynthesis was calculated as A + R (A = net photosynthesis; R = dark respiration). Photoinhibition was minimized by the addition of KHCO3 (non-limiting inorganic carbon source). Chlorophyll fluorescence measurements. Chlorophyll fluorescence measurements were made simultaneously with oxygen exchange measurements with a PAM 101 Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany). At the end of the dark period, a saturating light pulse (approximately 4,000 µmol m−2 s−1) (KL2500 LCD, Schott AG, Mainz, Germany) was applied over the measuring light to determine maximum efficiency of photosystem II (Fv/Fm), and another saturating pulse was given at the end of the light period to determine the effective efficiency of photosystem II (ΦPSII). Conductivity measurements. After oxygen evolution measurements, the solution from the electrode chamber was collected, diluted in 3 mL of deionised water and conductivity was measured with a conductimeter (Con 5 – EcoScan, Eutech Instruments, Singapore). Mitochondrial respiration inhibition experiments. Independent experiments were made to determine the effects of mitochondrial respiration inhibitors (0.03 mM myxothiazol and 0.3 mM propyl gallate) on oxygen consumption.

Results Oxygen exchange In the dark, control samples presented average values of oxygen consumption of 15 µmol O2 kg−1 DW s−1. After 30 min in a RH controlled atmosphere (23%, 50%, 95%), samples presented very high oxygen consumption in the dark for the first 5 min (Fig. 1), which was not inhibited by the


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Fig. 1 Oxygen consumption in the dark by Fontinalis antipyretica after several periods of drought stress (30 min up to 40 days) at different RH (23%, 50%, 95%)

Table 1 Inhibition of oxygen consumption of Fontinalis antipyretica by the mitochondrial respiration inhibitors myxothiazol and propyl gallate Oxygen consumption (µmol O2 kg−1 DW s−1)

Control Stressed samples (23% RH)

production was almost absent. In 95% RH, only 50% of the oxygen production was inhibited, and after 7 days there was still some degree of measurable oxygen production, although very low (Fig. 2C).

No inhibitors

Myxothiazol 0.03 mM + propyl gallate 0.3 mM

Inhibition (%)

Chlorophyll fluorescence

6.1 210

0 210

100 0

The average control value of Fv/Fm was 0.735. After 30 min at 23% RH, it decreased 25% being completely inhibited after 2 h (Fig. 2D). In 50% RH, the decrease of Fv/Fm was less rapid (Fig. 2E). In 95% RH, only a 5–10% decrease was observed after 24 h and of 60% after 1 week, being totally inhibited after 2 weeks (Fig. 2F). For 23% and 50% RH an inhibition of about 10% of ΦPSII was observed after 30 min of stress over the control value of 0.395. However, in the first case, the effective PSII photochemical efficiency decreased to 0 after 2 h (Fig. 2D), while in the second case an inhibition of only 40% had occurred (Fig. 2E). At 95% RH, an inhibition of 20% after 24 h and of 75% by the end of the week was observed (Fig. 2F). Total inhibition occurred after 2 weeks. These values are in accordance with the trend of gross photosynthesis.

mitochondrial respiration inhibitors myxothiazol and propyl gallate (Table 1). The increase was highest (about 1,000%) in RH 23%. After 30 min of desiccation, this increase in O2 consumption was already present. However, after 1 week, this value was about half of the observed maximum. Control samples presented an average value of gross photosynthesis of about 23 µmol O2 kg−1 DW s−1. After 30 min in 23% RH (Fig. 2A) and 50% RH (Fig. 2B), oxygen production decreased about 60% and 35%, respectively, and after 1 h oxygen

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Fig. 2 Gross photosynthesis (●) and chlorophyll fluorescence parameters Fv/Fm (°; —) and ΦPSII (∆; - - -) of Fontinalis antipyretica after several periods of drought stress (30 min up to 40 days) at different RH (A, D: 23% RH; B, E: 50% RH; C, F: 95% RH)

Conductivity A small increase in the conductivity was measured in the three tested conditions after 30 min.

However, with the increase of the stress period, a higher increase in the conductivity was observed presenting maximum values after 24 h in 23% HR and 50% HR (Table 2).


Table 2 Conductivity measurements of Fontinalis antipyretica after several periods of drought stress (30 min up to 40 days) at different RH (23%, 50%, 95%) Conductivity (µS cm−1 mg−1 DW)

0 min 30 min 60 min 120 min 24 h 7 days 14 days 40 days

23% RH

50% RH

95% RH

1.2 ± 0.8 5.3 ± 0.4 8.8 ± 1.9 10.4 ± 1.0 15.0 ± 1.3 15.4 ± 1.2 14.9 ± 1.1 15.3 ± 1.0

1.2 ± 0.8 2.6 ± 0.6 7.4 ± 2.2 7.7 ± 0.8 13.5 ± 1.1 16.6 ± 2.0 13.5 ± 0.9 15.2 ± 1.9

1.2 ± 0.8 3.6 ± 1.3 4.0 ± 0.9 2.0 ± 1.1 2.9 ± 0.6 9.1 ± 1.5 12.1a 9.7 ± 2.4

a

Only one observation

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extreme conditions. These results show the impact of water loss on photosystem II integrity in this aquatic bryophyte. Results with more desiccationtolerant species like Anomodon viticulosus showed recovery of Fv/Fm in 10 min after 8–10 days of desiccation (Proctor and Smirnoff 2000). Deltoro et al. (1998) found that species from hydric habitats were unable to resume photosynthesis, which could result from photoinhibition or membrane damage. In fact, F. antipyretica showed to be extremely sensitive to the imposed desiccation conditions being unable to recover its normal metabolic activity (Figs. 1 and 2). One of the suggested sites of damage might be the cellular membranes since a substantial electrolyte leakage was observed (Table 1).

Discussion In this work we found a substantial increase in the O2 consumption during the dark that was not suppressed by mitochondrial inhibitors. Other authors also have found that the initial burst of oxygen consumption after a stress period, was insensitive to mitochondrial respiration inhibitors, like myxothiazol and propyl gallate (Marré et al. 1998). Although there are many oxygen consuming reactions in the cell, namely in the glyoxysome, in the chloroplast (Mehler reaction) and in the plasma membrane (due to the activity of membrane NADPH oxidase) (Bhattacharjee 2005), that could explain this non-mitochondrial oxygen consumption, the magnitude of that phenomenon suggests that it may be due to a generalised non-specific oxidative process, possibly associated with the activity of polyphenol oxidases (Thipyapong et al. 2004). In fact, the loss of tonoplast integrity may bring the vacuolar content, enriched in phenols, in contact with cytosolic enzymes, namely polyphenol oxidases, leading to the oxidation of the former and to the measured oxygen consumption burst. The observed browning of the photosynthetic tissues supports this hypothesis. Gross photosynthesis was highly affected by desiccation, decreasing significantly with lower RH. This was in accordance with the decrease observed in the Fv/Fm, a measure of the maximum quantum efficiency of photosystem II, in the more

Acknowledgments. This work has been supported by Fundação para a Ciência e Tecnologia (FCT) grant BD/31424/2006, Lisbon, Portugal. Thanks to Rute Vieira for providing Fontinalis antipyretica specimens.

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