Journal of Experimental Botany, Vol. 51, No. 351, pp. 1695-1704,
October 2000
© 2000 Oxford University Press
Original Papers |
Rapid recovery of photosystems on rewetting desiccation-tolerant mosses: chlorophyll fluorescence and inhibitor experiments
School of Biological Sciences, University of Exeter, Hatherly Laboratories, Prince of Wales Road, Exeter EX4 4PS, UK
Received 16 December 1999; Accepted 2 June 2000
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Abstract |
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In the mosses Racomitrium lanuginosum, Anomodon viticulosus and Rhytidiadelphus loreus, after a few days air dry, Fv/Fm reached, within the first minute of remoistening in the dark, two-thirds or more of the value attained after 40 min. A fast initial phase of recovery was completed within 10–20 min after which further change was slow. Initial recovery of
PSII in the light was somewhat slower, but was generally substantially complete within a similar time. Remoistening with 0.3 mM cycloheximide (CHX) or 3 mM dithiothreitol (DTT) made little difference to this short-term (40 min) recovery of either Fv/Fm or PSII; 3 mM chloramphenicol (CMP) had little effect on recovery of Fv/Fm, but resulted in substantial (though not total) depression of PSII and 14CO2 uptake. Effects of the protein-synthesis inhibitors and DTT were much more clearly apparent in longer-term experiments (>20 h) but only in the light. In the dark, the three inhibitors had at most only slight effects over periods of 60–100 h. In the light, CMP-treated samples of all three species showed a progressive decline of dark-adapted Fv/Fm, falling to zero within 1–5 d (possibly due to blocking of the turnover of the D1 protein of PSII) and accelerated by DTT. CHX-treated samples showed a similar but slower decline. In the shade-adapted and relatively desiccation-sensitive Rhytidiadelphus loreus, slow recovery of Fv/Fm continued in the dark even in the presence of CMP and CHX for much of the 142 h of the experiment. The results indicate that in desiccation-tolerant bryophytes recovery of photosynthesis after periods of a few days air dry requires only limited chloroplast protein synthesis and is substantially independent of protein synthesis in the cytoplasm. Key words: Anomodon viticulosus, bryophytes, desiccation tolerance, protein synthesis, Rhytidiadelphus loreus, Racomitrium lanuginosum.
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Introduction |
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Desiccation tolerance in bryophytes has an extensive literature (reviewed by Bewley, 1979
; Bewley and Krochko, 1982; Proctor, 1981, 1982, 1990; Oliver and Bewley, 1997). Much of the earlier work sought simply to establish the facts of desiccation tolerance—how long the plants would survive in the dry state and what equilibrium humidity they would tolerate (Clausen, 1952; Abel, 1956; Hosokawa and Kubota, 1957; Dilks and Proctor, 1974; Schonbeck and Bewley, 1981). In more recent decades emphasis has moved to the basis and mechanisms of tolerance, including the possible role of sugars and other carbohydrates (Smirnoff, 1992; Marschall et al., 1998), and of antioxidant systems (Dhindsa and Matowe, 1981; Dhindsa, 1987; Seel et al., 1991, 1992a, b; Smirnoff, 1993).
Several authors have particularly emphasized the importance of protein synthesis (Bewley, 1972, 1973, 1979; Bewley and Krochko, 1982; Bewley and Oliver, 1992; Oliver, 1991, 1996; Oliver and Bewley, 1997). They have suggested that in fully desiccation-tolerant plants (exemplified by the moss Tortula ruralis), recovery depends on a combination of constitutive tolerance, and ‘repair’ which takes place during and following rehydration. In ‘modified’ desiccation-tolerant plants (including many vascular ‘resurrection plants’) tolerance depends on protective mechanisms (commonly ABA-induced) set in place during slow drying, with (in different species) more or less extensive repair of cell damage following rehydration (Oliver et al., 1998). Recent gas exchange and chlorophyll fluorescence measurements have shown that recovery of photosynthesis in bryophytes after desiccation can be remarkably rapid, and appears to leave little time for repair processes requiring synthesis of proteins or other cell components on any substantial scale (Tuba et al., 1996; Csintalan et al., 1999; Marschall and Proctor, 1999).
The present paper examines the rate of recovery of normal photosynthetic function in some representative mosses, largely using chlorophyll fluorescence measurements, and the effect of inhibitors of chloroplast and cytoplasmic protein synthesis (Bottomley and Bohnert, 1982; Galling, 1982; Dace et al., 1998) on immediate and longer-term recovery. It addresses three questions. First, is the initial recovery of photosynthesis in bryophytes after desiccation dependent on protein synthesis? Second, what is the effect of light on recovery from desiccation, and is photoprotection significant to the course of recovery? Third, at what point, and under what conditions, does protein synthesis become a significant factor in the recovery process?
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Materials and methods |
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Plant material and experimental procedure
Three moss species were used in the experiments. Anomodon viticulosus (Hedw.) Hook. and Tayl. was collected from a near-vertical Devonian limestone rock face in woodland at Chudleigh, Devon (National grid reference SX 865 786; Lat. 50° 36' N, Long. 3° 36' W)). Racomitrium lanuginosum (Hedw.) Brid. was collected from granite field walls near the O Brook, Holne, Dartmoor, Devon (SX 665 720; 50° 32' N, 3° 53' W). Rhytidiadelphus loreus (Hedw.) Warnst. was collected in rocky Quercus petraea woodland near Bench Tor, Holne, Dartmoor (SX 689 721; 50° 32' N, 3° 51' W). All were maintained, as needed, either dry in a cold room at 5 °C, or fully hydrated in polythene bags in a north-facing laboratory window. The laboratory atmosphere, which varied little from 20 °C, 60–70% r.h. (
-50 to -70 MPa) over the period of our measurements, was used as a standard drying environment. If measurements at the end of a drying period had to be spread over two or more days, the material was kept in the cold room at 5 °C between measurements to minimize the effect of the additional desiccation time (Hearnshaw and Proctor, 1983).
Chlorophyll fluorescence measurements
Fluorescence measurements were made using a Hansatech FMS1 modulated fluorometer (Hansatech Instruments, King's Lynn, UK). The abbreviations for chlorophyll fluorescence parameters and their derivation are as described previously (Schreiber et al., 1995). The modulated beam was checked with all three species to ensure that it was set at a level that would have no photochemical effect and the high intensity light pulses (8000 µmol m-2 s-1) were saturating. Light response curves of relative electron transfer rate and NPQ for the three species were constructed using the scripting facility on the FMS1. After an initial dark-adapted measurement of Fv/Fm, the mosses were exposed to a sequence of increasing actinic light levels. After 5 min at each level, F and 
were measured for calculation of PSII, relative electron flow and NPQ.
For the short-term recovery experiments, two parallel sets of three matched samples were taken, one set remoistened and recovering in darkness, and the other with actinic light provided by the fluorometer (50 or 100 µmol m-2 s-1 PPFD, depending on species). The shoot samples were placed dry in standard Hansatech leaf clips, and remoistened with 200 mm3 of water or inhibitor solution applied by pipette. Saturating flashes 1, 3, 5, 7 10, 15, 20, 25, 30, and 40 min after remoistening provided measurements of Fv/Fm or PSII, respectively, for the ‘dark’ and ‘light’ samples.
For the long-term recovery experiments the dry samples were placed on filter papers in 9 cm plastic Petri dishes, and remoistened by pouring on water or inhibitor solution. One set of replicates was kept dark, and the other at c. 125 µmol m-2 s-1 PPFD under a bank of fluorescent tubes. At appropriate intervals the shoots were loaded into leaf clips to dark adapt and, following measurement of Fv/Fm, returned to their Petri dishes.
Metabolic inhibitors
Dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (Winter and Königer, 1989), was used at 3 mM, which appeared to give reasonably complete suppression of xanthophyll-mediated NPQ without undue side effects. Chloramphenicol (CMP) was used as an inhibitor of chloroplast protein synthesis at 3 mM, and cycloheximide (CHX) as an inhibitor of cytoplasmic protein synthesis at 0.3 mM. The effect of the last two was checked by assaying the incorporation of 14C-leucine into proteins by Anomodon shoots, in the dark and in light at 125 µmol m-2 s-1 PPFD.
Incorporation of L-[1-14C]leucine into proteins
Samples of fully hydrated Anomodon viticulosus shoots were blotted free of excess water, their fresh weight (c. 75 mg) recorded, allowed to dry at laboratory humidity and temperature for 3 d, and then kept dry at 5 °C for 5 weeks. The samples were placed dry into glass vials, and remoistened by pipetting onto each 500 mm3 of water (control) or inhibitor solution containing 14.8 kBq [1-14C]leucine (2.09 GBq mmol-1). The inhibitor treatments were applied in a 2x2 factorial design with one set of three replicates incubated for 20 min in light (125 µmol m-2 s-1 PPFD) and another set in the dark. The moss samples were removed from the vials, rinsed and blotted, and placed in Eppendorf tubes in liquid nitrogen, stored frozen overnight, and extracted in 1 cm3 50 mM HEPES at pH 7.0. The tubes were centrifuged, and a 0.5 cm3 sample of the supernatant was transferred to a clean Eppendorf and proteins precipitated by adding 0.5 cm3 20% (w/v) trichloroacetic acid (TCA). After standing for 10 min the tubes were centrifuged. The pellets were twice resuspended in 0.5 cm3 10% TCA and centrifuged. The final washed pellets were resuspended in 0.5 cm3 water and transferred in toto to vials with 4 cm3 scintillation fluid (Tri-Carb 2500 TR, Packard) and the 14C content determined by liquid scintillation counting.
For a more precise assessment of the effect of the inhibitor solutions at faster rates of protein synthesis, more material and a longer exposure time, samples of fully hydrated moss shoots, c. 200 mg FW, were placed in glass vials with 500 mm3 of water (control) or inhibitor solution, and maintained for 2 h either in the dark or under a bank of fluorescent tubes at 75 µmol m-2 s-1 PPFD. To each vial was then added 4 mm3 [1-14C]leucine (7.4 kBq, 2.09 GBq mmol-1) per sample, and incubation was continued for a further 2 h under the same conditions. Treatment then continued as above.
Photosynthetic 14CO2 uptake
Samples of cut, fully hydrated moss shoots 20 mm in length were blotted, weighed, and allowed to dry as for the preceding experiments. The shoots were placed in flat-bottomed 25 mm diam glass vials under a bank of fluorescent tubes giving 125 µmol m-2 s-1 PPFD. At the start of the experiment 0.5 cm3 of water or inhibitor solution was added to all the vials by pipette. For the time-course of Fig. 7, 10 mm3 of label solution containing 18.5 kBq Na214CO2 was added to individual vials at appropriate intervals, followed after 4 min by 0.5 cm3 glacial acetic acid. For the factorial experiment of Fig. 8 label was added to all the samples after 1 h. The acid-stable 14C content of an aliquot of the sample was then determined by liquid scintillation counting as described above. In these experiments more than 90% of the label was in soluble form and dark 14CO2 fixation was less than 10% of light fixation.
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Results |
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Some background measurements
PPFD response curves of relative electron flow rate and NPQ for fully hydrated material of the three species used in the recovery experiments are presented in Fig. 1
. Rhytidiadelphus loreus is markedly shade adapted and Anomodon viticulosus only a little less so. As would be expected from its habitat, photosynthetic electron flow in Racomitrium lanuginosum continues to increase to much higher PPFD levels. Even the shade-tolerant R. loreus shows substantial NPQ, but this is saturated at relatively low PPFD levels. A. viticulosus shows rather higher NPQ (consistent with its greater light tolerance). The massively high NPQ seen in R. lanuginosum (far above the levels found in vascular plants) seems to be a common characteristic of mosses in open sun-exposed habitats; similarly high values have been measured in Andreaea rothii, Aulacomnium palustre, Schistidium apocarpum, Scorpidium scorpioides, Sphagnum cuspidatum, Tortula intermedia, T. ruralis, and Ulota crispa. NPQ in all three of the present species is very largely suppressed by DTT. This inhibitor also depresses the relative electron flow curve, slightly in R. loreus and A. viticulosus, and dramatically in R. lanuginosum—the combined effect on PSII, NPQ and qP (not shown) virtually turns the responses of this species into those of a shade plant.
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Measurements of the incorporation of 14C-leucine into TCA-insoluble proteins by shoots of A. viticulosus are summarized in Fig. 2
. During the first 20 min of rewetting, the effect of CMP was highly significant (P<0.001), reducing leucine incorporation to around 25% of control (Fig. 2a). Other treatments are not statistically significant, but the means suggest somewhat higher rates of protein synthesis in the light, and that CHX suppresses much of the protein synthesis unaffected by CMP. In fully hydrated material (Fig. 2b) the effects of both CMP and CHX are highly significant (P<0.001). In the presence of both CMP and CHX protein synthesis is reduced to low levels; different experiments gave figures varying between 4% and 14% of control. Light has no significant effect (P=0.350), but there is a significant lightxCHX interaction (P=0.023), possibly related to the rapid light-induced synthesis and turnover of the PSII D1 protein, uninhibited in the CHX treatment.
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Immediate recovery from desiccation: the first 40 min
The immediately striking feature of the recovery curves of the mosses on rewetting after a few days air dry is the remarkably rapid rise of Fv/Fm on remoistening in the dark, reaching within the first minute two-thirds or more of the value attained after 40 min (Figs 3–6). Just how rapid the early stages of recovery can be is shown for Anomodon viticulosus in Fig. 3. In this case the fluorescence level of the dry moss is comparable with that after rewetting, and there is measurable variable fluorescence, giving a mean Fv/Fm just under 0.04; as the moss takes up water, Fm immediately rises, and F0 falls. Generally, the fast initial rise of Fv/Fm was completed within 10–20 min, after which further change was slow. In Anomodon viticulosus remoistened after 8 d dry (Fig. 4a), Fv/Fm returned to near-normal values (>0.7) within 15 min. In material of the same species kept dry for 40 d (Fig. 4b) the values of Fv/Fm 1 min after remoistening were only 0.4–0.5. Subsequent recovery was slow and somewhat variable, reaching values averaging about 0.65 after 24 h. In Racomitrium lanuginosum after 10 d dry (Fig. 5) initial recovery was rather slower. This may have been partly due to slower rehydration of the shoots, although that seems unlikely to be the whole explanation. Nevertheless, Fv/Fm reached 0.4–0.5 within the first minute, and after 20 min had attained relatively steady values around 0.7. Rhytidiadelphus loreus was more sensitive to desiccation than either of the preceding species, but freshly-collected material still showed rapid recovery to values around 0.6 after 3 d air dry (Csintalan et al., 1999); material dried after being maintained in a saturated atmosphere for several weeks was more sensitive, Fv/Fm reaching only 0.45–0.50 within the first minute (Fig. 6).
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0.001***.
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Remoistening with 3 mM CMP, 0.3 mM CHX or 3 mM DTT instead of water made relatively little difference to the course of recovery of Fv/Fm in any of the three species. The apparently large difference between the Fv/Fm curves for CMP and CHX and the water control in R. loreus depends mostly on one of the three replicates in each case.
Recovery of PSII in the light tended to be somewhat slower, but after short periods dry was generally substantially complete within 10–15 min. Again, the effect of inhibitors was generally not great. There appears to be a significant depression of PSII by CMP in A. viticulosus desiccated for 8 d, but this is not seen in material that had been dry for 40 d. There was also a slight apparent depression of PSII in R. lanuginosum by CMP+DTT (not shown). However, in all of the experiments PSII recovered in the course of 40 min to 75% or more of the control mean regardless of the presence of inhibitors.
Effects on 14CO2 fixation following rehydration
The rate of 14CO2 fixation in Anomodon viticulosus recovered more slowly than PSII, and recovery appeared still not fully complete after 2 h; CMP+CHX depressed PSII by about 25% (in both light and dark) and 14CO2 fixation by about 50% over this period (Fig. 7
). The data for 14CO2 fixation are very variable, but there is some indication that the inhibitors may have had a particularly marked effect from about 5–20 min after remoistening. The factorial experiment of Fig. 8 shows a highly significant effect of CMP on 14CO2 fixation in all three species, but no effect of CHX. The effect of CMP follows the desiccation sensitivity of the species, least in Racomitrium lanuginosum and greatest in Rhytidiadelphus loreus.
Longer-term changes
More dramatic effects of protein-synthesis inhibitors appeared only in experiments extending over a few days, and then only in light. In A. viticulosus, remoistened and maintained in the dark after 10 d dry (Fig. 9), dark-adapted Fv/Fm rose rapidly on remoistening to between 0.7 and 0.8 and remained unchanged for 60 h; material treated with CMP followed a similar course at slightly lower Fv/Fm, closely paralleled by untreated, control material remoistened and maintained in the light. DTT-treated material recovering in the dark showed a slight progressive decline of Fv/Fm from 30 h onwards, and the CMP+DTT treatment a somewhat steeper decline reaching Fv/Fm0.6 after 60 h.
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The really striking effects were shown by the inhibitor treatments in light. In A. viticulosus, the DTT-treated samples showed a steep initial decline of dark-adapted Fv/Fm, with some indication of levelling off at Fv/Fm
0.3 after 30–60 h. The CMP-treated samples showed a steep progressive decline, Fv/Fm falling to less than 0.1 after 55 h. This decline was even faster and steeper in material treated with CMP+DTT. CHX-treated samples showed a similarly progressive but slower decline, Fv/Fm falling to 0.25 after 95 h. The pattern in R. lanuginosum was essentially similar (Fig. 10). The main difference was the much less marked effect of CHX in the light, Fv/Fm declining only to 0.7 after 55 h. In the presence of CMP+DTT, Fv/Fm fell to zero within 55 h.
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Rhytidiadelphus loreus, the most desiccation sensitive of the three species and quite highly stressed after 3 d in the dry state, showed a broadly similar pattern with some significant differences (Fig. 11
). One hour after remoistening, mean Fv/Fm for all treatments was between 0.45 and 0.55. From this point Fv/Fm in all the ‘dark’ treatments increased, quickly at first and then more slowly, the control samples settling between 0.75 and 0.8 after 50–60 h, and the inhibitor treatments following a roughly parallel course c. 0.05 units lower. Fv/Fm of the ‘light’ control samples rose, but more gradually, running for the first 50 h at about 0.2 units below the ‘dark’ control, then converging to within 0.1 unit of that. In the DTT treatment Fv/Fm remained little changed throughout at 0.5. In the CMP treatment, Fv/Fm had fallen almost to zero after 142 h. In the CHX treatment a temporary rise of Fv/Fm in the first 20 h was followed by a long progressive decline to a final value of c. 0.2.
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Sources of error and the reliability of conclusions
There are hazards of interpretation in both chlorophyll fluorescence measurements and inhibitor experiments, and inferences from them are potentially open to criticism. The most widely used fluorescence parameters are ratios, especially Fv/Fm, (
-F)/ (PSII) and (Fm-)/ (NPQ). Fv/Fm, (on which much of this argument rests) should be relatively insensitive to moderate variations in light absorption by the plant material (which could be caused for example by small movements of the tissue during rehydration), and all three should be independent of the absolute amount of chlorophyll present. All our measurements (except those on dry material in Fig. 3) were made in the presence of excess water, so would always have been on the level plateau of light-absorbance/water-content curves of the kind presented by Seel et al. (Seel et al., 1992c). It is possible in principle for a fully-recovered Fv/Fm to reflect full recovery of a part of the material, and complete degradation of the chlorophyll in the remainder, but there is no visual indication that this has occurred in these experiments. From preliminary tests and scrutiny of the experimental results, CMP, CHX and DTT at the concentrations used appeared to act as effective inhibitors, but not to have side-effects that could compromise the results. None showed more than slight effects on recovery of Fv/Fm in the dark over periods of several days. In fully-hydrated material, CMP and CHX suppressed c. 90% of protein synthesis. In the first 20 min of recovery, CMP suppressed c. 75% of protein synthesis. CHX appeared to suppress about half of the remainder. DTT was clearly effective in suppressing NPQ in the light-response measurements and had no serious side effects on tissue kept in the dark.
Recovery of photosynthesis during rehydration
The clearest conclusion to emerge from the present results is that the photosystems of desiccation-tolerant mosses survive a drying and rewetting episode essentially intact, and return to a functional state with remarkable rapidity. Alongside that, it is also clear that there are other components which recover more slowly, and some that require at least some protein synthesis for full normal recovery.
The remoistening experiments demonstrated unequivocally that CMP and CHX at concentrations shown to reduce protein synthesis to low levels had little or no effect on the initial recovery of the photosystems (as measured by chlorophyll fluorescence) either in the dark or in the light. This is consistent with the rapid initial recovery of Fv/Fm and PSII seen in all three of our species, and in Grimmia pulvinata (Csintalan et al., 1999). It is also consistent with the rapid recovery shown in the fluorescence measurements of Marschall and Proctor on the leafy liverwort Porella platyphylla (Marschall and Proctor, 1999), and in the slow-fluorescence and gas-exchange measurements of Csintalan et al. on the desiccation-tolerant moss Tortula ruralis (Csintalan et al., 1999). In the present experiments CMP and CHX had virtually no effect on dark recovery, even over a period of several days during which they showed strongly marked and characteristic effects in the light. In the case of DTT, it is certain that the inhibitor penetrates the cells within minutes in sufficient concentration to suppress NPQ (Csintalan et al., 1999). It is noteworthy that not only the rapid initial recovery, but also the continuing rise of Fv/Fm in R. loreus over the ensuing 50 h is apparently insensitive to protein-synthesis inhibitors. However, the measurements presented in Figs 7 and 8 indicate that some organelle-encoded protein synthesis early in the course of recovery is needed for return to normal rates of CO2 uptake. This hints at desiccation sensitivity (and perhaps rapid turnover) of one or more components of photosynthesis that affect CO2 fixation more than electron transport. Because the interactions and feedback between photochemistry, Calvin cycle activity and metabolism in the cytosol and mitochondria during photosynthesis are complex it not possible to do more than speculate on the cause of slower recovery of CO2 fixation and its sensitivity to CMP. The ‘rehydrins’ expressed following rehydration of the moss Tortula ruralis (Oliver, 1991, 1996; Oliver et al., 1998) could possibly be involved. Physiological characterization of sites of damage combined with the development of EST libraries for moss species (Machuca et al., 1999; Wood et al., 1999) should in due course allow identification of the relevant proteins.
In this paper, the rapidity of recovery of photosynthesis of desiccation-tolerant mosses has been particularly emphasized. However, it must also be emphasized that the rate of recovery varies greatly between species; some difference is apparent in the three considered here. In general, recovery is fast in species of exposed situations that experience rapid alternations between wet and dry conditions (e.g. Tortula (Syntrichia) ruralis, Grimmia pulvinata), but may be much slower in woodland species (e.g. Mnium hornum, Polytrichum formosum) subject to less frequent changes, but which may, however, be able to survive prolonged drying.
Relationship of photosynthesis to repair processes during recovery from desiccation
The results discussed above suggest that processes related to thylakoid function recover quickly and are little affected by protein synthesis inhibitors, while recovery of 14CO2 fixation is somewhat slower and more-dependent on organellar protein synthesis. From diverse evidence, including electron micrographs of cells in freshly-rehydrated moss leaves (reviewed by Oliver and Bewley, 1984) and electrolyte leakage following rehydration, Oliver and his co-workers (Oliver, 1991, 1996; Bewley and Oliver, 1992; Oliver and Bewley, 1997; Oliver et al., 1998) have regarded desiccation tolerance in bryophytes as essentially repair based, and have linked this particularly with the synthesis of the characteristic proteins (rehydrins) preferentially synthesized following rehydration. Repair clearly must embrace a diverse range of processes with widely-different time courses, from reimbibition of cell walls and reinstatement of water into macromolecules (likely to be largely completed within minutes) to expression of the rehydrins which may extend over a few hours. Analysis of this diversity of processes is a major challenge for future research.
It has been demonstrated that ribosomes of Tortula ruralis maintain their integrity following desiccation (Bewley, 1972), and protein synthesis recommences within minutes of rehydration (Gwózdz et al., 1974; Oliver, 1991; Oliver et al., 1993). The present results show that the photosystems and almost certainly much of the rest of the photosynthetic system is similarly retained intact. If the photosynthetic and protein-synthesis machinery of the cell can survive a desiccation episode essentially intact and functional, there is no a priori reason why other major metabolic systems should not survive similarly. If this is so, then much of the repair process is likely to be a matter of reassembly rather than resynthesis; this seems likely also from considerations of energetics and synthesis capacity. The speed of return to pre-desiccation rates of gas exchange in manometric and IRGA measurements (Hinshiri and Proctor, 1971; Dilks and Proctor, 1974, 1976; Tuba et al., 1996) also argues for the survival substantially intact of the major metabolic systems that affect carbon balance. There is certainly leakage of electrolytes immediately on remoistening, but normal membrane integrity appears to be re-established quickly in desiccation-tolerant species (Deltoro et al., 1998). None of this should imply that protein synthesis is unimportant in recovery. Even if it is quantitatively modest in relation to the total protein complement of the cells, it may still be vitally important to diverse systems and processes. These results show that chloroplast protein synthesis significantly affects recovery of at least one component of the photosynthetic system, and the rehydrins may be crucial in the full return of the cell to the pre-desiccation state, and perhaps to the drought hardening observed even in highly desiccation-tolerant species (Abel, 1956; Dilks and Proctor, 1976; Schonbeck and Bewley, 1981). In the poikilochlorophyllous desiccation-tolerant flowering plant Xerophyta humilis, it was found that initial recovery was virtually independent of de novo transcription of nuclear genes, but that it did require synthesis of new proteins (Dace et al., 1998). In the pteridophyte Selaginella lepidophylla, reassembly of a normal photosynthetic apparatus proceeds on the addition of water in the dark, even in the presence of CMP, which only becomes inhibitory to PSII function following illumination (Eickmeier et al., 1993; Osmond, 1994). The relation of the rate of recovery of photosynthesis to the apparent degree of organelle damage seen in electron micrographs of freshly-rehydrated material of desiccation-tolerant bryophytes awaits further critical examination and resolution.
Interactions with other processes
At what point and under what conditions does protein synthesis become critically important? In light, on a scale of hours, CMP brings about a marked and progressive decline in Fv/Fm, reducing photosynthetic activity to zero within a day or two even at the relatively low irradiance that was used here, and this effect is reinforced by DTT. This suggests that what is probably being seen is the effects of the fast turnover in light of the plastid-encoded D1 protein of PSII (Anderson et al., 1997), the rate of destruction of the protein increasing when the photoprotection mediated by the xanthophyll cycle is suppressed by DTT. CHX has a less immediate and less drastic effect. The ‘light’ control and CHX curves for Fv/Fm only diverge after about the first 20 h, following which the CHX curve shows a progressive slow decline while the ‘light’ control shows a slow recovery towards its ‘dark’ counterpart. This effect is apparent in all three species, but is least marked in R. lanuginosum.
Taken as a whole, the results suggest that much of the requirement for protein synthesis during recovery of desiccation-tolerant bryophytes (and, indeed, during their normal metabolism) is brought about by photo-oxidative damage (Foyer et al., 1994), perhaps especially to the PSII D1 protein (Nicklesen and Rochaix, 1994; Ohad et al., 1994; Anderson et al., 1997). Synthesis of proteins, particularly those involved in repair and antioxidant processes, is needed to maintain function in the light, but is not a major requirement for the initial rapid recovery of photosynthesis after rehydration. The results also underline that non-photochemical quenching and xanthophyll-cycle dependent photoprotection (Björkman and Demmig-Adams, 1995; Horton et al., 1996; Eskling et al., 1997; Gilmore, 1997) are likely to be of particular importance to mosses.
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Acknowledgments |
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We thank Marjorie Raymond for excellent technical assistance, and an anonymous referee for perceptive and helpful comments.
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1 To whom correspondence should be addressed. Fax: +44 1392 263700. E-mail:M.C.F.Proctor{at}exeter.ac.uk
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References |
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