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Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2003? 2003 26?12751286 Original Article Desiccation and light stress J. M. Farrant et al. Plant, Cell and Environment (2003) 26, 1275–1286 An investigation into the role of light during desiccation of three angiosperm resurrection plants J. M. FARRANT1, C. VANDER WILLIGEN1,2 D. A. LOFFELL2, S. BARTSCH2 & A. WHITTAKER1 1 Department of Molecular and Cell Biology and 2Department of Botany, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa ABSTRACT Under water-limiting conditions excitation energy harnessed by chlorophyll can lead to the formation of reactive oxygen species (ROS). Resurrection plants minimize their formation by preventing the opportunity for light–chlorophyll interaction but also quench them via antioxidants. Poikilochlorohyllous species such as Xerophyta humilis break down chlorophyll to avoid ROS formation. Homoiochlorophyllous types retain chlorophyll. We proposed that leaf folding during drying of Craterostigma wilmsii and Myrothamnus flabellifolius shades chlorophyll to avoid ROS (Farrant, Plant Ecology 151, 29–39, 2000). This was tested by preventing leaf folding during drying in light. As controls, plants were dried without light, and X. humilis was included. Craterostigma wilmsii did not survive drying in light if the leaves were prevented from folding, despite protection from increased anthocyanin and sucrose and elevated antioxidant enzyme activity. Membranes were damaged, electrolyte leakage was elevated and plastoglobuli (evidence of light stress) accumulated in chloroplasts. Restrained leaves of M. flabellifolius survived drying in light. Leaf folding allows less shading, but the extent of chemical protection (anthocyanin content and antioxidant activity) is considerably higher in this species compared with C. wilmsii. Chemical protection appears to be light regulated in M. flabellifolius but not in C. wilmsii. Drying in the dark resulted in loss of viability in the homoiochlorophyllous but not the poikilochlorophyllous species. It is hypothesized that some of the genes required for protection are light regulated in the former. Key-words: Craterostigma wilmsii; Myrothamnus flabellifolius; Xerophyta humilis; desiccation tolerance; homoiochlorophyllous; leaf folding; light stress; poikilochlorophyllous. INTRODUCTION Resurrection plants are able to survive desiccation to the air-dry state. Bewley (1979) has proposed that there are three criteria which must be met for plant tissue to survive severe loss of protoplasmic water: It must: (1) limit damage Correspondence: Jill M. Farrant. E-mail: [email protected] © 2003 Blackwell Publishing Ltd incurred to a repairable level; (2) maintain its physiological integrity in the dried state; and (3) mobilize repair mechanisms upon rehydration which effect restitution of damage suffered during desiccation. Subsequent reports (for example Bewley & Oliver 1992; Oliver, Woods & Mahony 1998) have suggested that desiccation tolerance is achieved either by mostly repair of desiccation- (and/or rehydration-) induced damage, or by of protection of cellular integrity during drying such that little by way of repair is required. Furthermore, it is generally held that the lower order resurrection plants (lichens, algae and bryophytes) plants achieve tolerance using the former mechanism and that angiosperms tend to protect against desiccation damage (Gaff 1989; Oliver & Bewley 1997; Oliver et al. 1998; Farrant et al. 1999; Farrant 2000; Cooper & Farrant 2002; Walters et al. 2002). There are a number of stresses brought about by, or in association with, extreme water loss (reviewed by Gaff 1989; Vertucci & Farrant 1995; Oliver et al. 1998; Farrant 2000; Walters et al. 2002), which must be protected against in angiosperm tissues while they are drying. One of the associated stresses in photosynthetically active tissues, is light. Under water-limiting conditions, excitation energy harnessed by chlorophyll cannot be dissipated via photosynthesis and can lead to the formation of oxygen free radicals, which, if left unquenched, can cause considerable subcellular damage (Halliwell 1987; Kaiser 1987; Larson 1988; Smirnoff 1993). The angiosperm resurrection plants appear to have a number of mechanisms, which vary among species, to minimize photo-oxidative damage. In all species examined to date, there is up-regulation of antioxidant systems in leaf tissues during drying which argues for some amelioration of free radical damage during dehydration and on subsequent rehydration (Smirnoff 1993; Sgherri et al. 1994a, b; Kranner & Grill 1997; Navari-Izzo et al. 1997a; Navari-Izzo, Quartacci & Sgherri 1997b; Sherwin & Farrant 1998; Farrant 2000). In addition to photo-chemical protection, resurrection plants undergo anatomical changes to prevent light–chlorophyll interaction and thereby put a stasis on photosynthesis and photo-oxidation reactions. Some species degrade chlorophyll and dismantle thylakoid membranes during dehydration. These plants, examples of which are the Xerophyta spp., Borya nitidia and Eragrostis nindensis are termed poikilochlorophyllous (Hallum & Luff 1275 1276 J. M. Farrant et al. 1980; Hetherington, Hallum & Smillie 1982; Tuba et al. 1996, 1997; Sherwin & Farrant 1998; Tuba, Proctor & Csintalan 1998; Farrant 2000; Vander Willigen et al. 2001). Many other species (e.g. Boea hydroscopica, Craterostigma spp., Myrothamnus flabellifolius and Sporobolus stapfianus) retain most of their chlorophyll and thylakoid integrity during drying (termed homoiochlorophyllous) (Sherwin & Farrant 1996, 1998; Quartacci et al. 1997; Farrant et al. 1999; Farrant 2000). Leaves of C. wilmsii and M. flabellifolius fold during drying causing shading of the inner surfaces from light. Those surfaces which are left exposed to light accumulate high levels of anthocyanins (Farrant 2000). These pigments mask chlorophyll and reflect photosynthetically active light, and also act as antioxidants (Larson 1988; Smirnoff 1993).We have proposed that these homoiochlorophyllous plants use leaf folding and anthocyanin accumulation as a means of minimizing light–chlorophyll interaction and thus photo-oxidative damage (Sherwin & Farrant 1998; Farrant 2000). The aim of this study was to determine whether leaf folding does indeed protect Craterostigma wilmsii Engl. and Myrothamnus flabellifolius Welw. from light stress during drying. Leaves were tied down in order to prevent natural folding during drying and the effect of this on plant viability was assessed using a number of physiological parameters. In order to determine whether the data obtained were due to light stress and not a consequence of restraining the leaves, plants with and without leaf restraints were also dried in the dark. As a further control to check for the effect of desiccation in the dark, the poikilochlorophyllous species Xerophyta humilis (Bak.) Dur. and Schinz, which does not significantly fold leaves, was also included in the study. MATERIALS AND METHODS Plant material and treatments during dehydration and rehydration Plants were collected and maintained in a glasshouse as previously described (Sherwin & Farrant 1996; Dace et al. 1998) until initiation of the experiments described below, which were performed under laboratory conditions. Plants were fully hydrated when the restraints were applied and were allowed to dry naturally [photosynthetic active radiation (PAR) = 400 mmol m-2 s-1) or in the dark by withholding water. In order to keep drying rates of plants dried in the light and dark comparable and to minimize time spent in the dark, plants were placed in the dark only when the soil had dried to 0.12 g H2O g-1 dry mass (< -1 MPa) but the plant tissues themselves were still at 100% relative water content (RWC). Drying to the air-dry state (approximately 5% RWC) took approximately 10 d for C. wilmsii and X. humilis, the period spent in the dark was only 5 d. Myrothamnus flabellifolius twigs dried within 48 h (light and dark treatments). Plants were maintained in the dry state for 1 week before rehydration. In all treatments plants were rehydrated in the light. Plants were sampled after 48 h rehydration as previous studies have shown that by this stage the tissues were fully rehydrated (100% RWC) (Farrant et al. 1999). The leaves of C. wilmsii grow in a rosette. During natural drying, the outer ring of leaves from the rosette fold over and shade the inner ones, the abaxial surfaces of the outer leaves being the only surfaces to receive light while the plant is in the dry state (Sherwin & Farrant 1998; Farrant 2000). For each experiment half of the plants in a tray were subject to leaf restraints and the other half were not. The outer leaves of the rosette were restrained by tying them to the soil with thin plastic (Parafilm; American National Can, Menasha, WI, USA) strips. Myrothamnus flabellifolius is a woody shrub and in the hydrated state the leaves are held perpendicular to the stem. During drying leaves fold upward through 90∞ becoming parallel to the stem axis with only the abaxial surface being exposed to light when in the dry state (Farrant 2000). Since it was difficult to restrain leaves from intact plants, detached branches of more than 10 cm length were used. Excised twigs and leaves of this species undergo normal leaf folding during drying and survive desiccation (Gaff & Loveys 1984; Farrant et al. 1999). Individual leaves from half of the branches which were allowed to dry were prevented from folding toward the stem by stapling across the blade (leaves are small and were not punctured). The twigs were rehydrated by placing cut ends into water. The leaves were sampled for the measurements outlined below from fully hydrated plants prior to drying, from the variously treated dry (< 5% RWC) plants and upon rehydration of these plants. The RWC was determined as previously described (Sherwin & Farrant 1996). The following codes were used to denote the various drying treatments: LU, plants dried in the light (L) the leaves unrestrained (U); LR, plants dried in the light with leaves restrained (R) from folding; DU, plants dried in the dark (D); leaves unrestrained; DR, plants dried in the dark, leaves restrained. Electrolyte leakage Membrane integrity of leaves from the variously treated plants was assessed by measuring the extent of electrolyte leakage from the tissue as described previously (Farrant et al. 1999). Five replicates of individual leaves of each of the species were used per treatment. Following each measurement the maximum leakage of the tissue was determined by snap freezing and thawing the leaves. The results are expressed at a percentage of maximum. Pigment analysis The chlorophyll and carotenoids were extracted from three replicates of leaf tissue from each of the the variously treated plants according to Lichtenthaler (1987). Material was cut into fine segments and pigments extracted in 100% acetone at 4 ∞C in the dark. Pigment concentrations in the supernatant was determined spectrophotmetrically (DU 650; Beckman, Fullerton, CA, USA) at 661.6, 644.8 and © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 Desiccation and light stress 1277 470 nm. Chlorophyll (a + b) and carotenoid (x + c) contents were calculated using extinction coefficients provided by Lichtenthaler (1987). Total anthocyanin content of leaves (three replicates per treatment) were determined as described in Sims & Gamon (2002). Extraction was in acidified methanol and the pigment in the supernatant determined spectrophotmetrically at 530 and 657 nm. Sucrose analysis Leaves from various treatments were ground in liquid nitrogen and sugars were extracted in 100 mM NaOH in 50% ethanol. The extract was neutralized by addition of 100 mM HEPES in 100 mM acetic acid, centrifuged at 16 000 g for 20 min and the extract retained. The pellet was re-extracted using the above procedure, and supernatants combined. Quantitation of sucrose was by use of the Dglucose/D-fructose sugar assay kit (Boehringer Mannheim, Mannheim, Germany) based on the methodology of Bergmeyer & Bernt 1974). The production of NADPH was determined spectrophotometrically at 340 nm (Beckman DU 6500) and used to calculate the quantity of sucrose in each sample. Antioxidant enzyme assays Our previous studies (Farrant 2000; Sherwin & Farrant 1998) have shown that ascorbate peroxidase activity (AP) increased markedly in all three species, and glutathione reductase (GR) activity increased in M. flabellifolius and X. humilis, during drying. Thus the activities of these antioxidant enzymes were measured in leaves of the variously treated plants. Leaf material (0.04–0.07 g) was ground in a mortar with liquid nitrogen. Insoluble polyvinylpyrrolidone (twice the weight of leaf material) was added and the enzyme extracted in 25 mM KH2PO4 (pH 7.5), 1 mM ethylenediaminetetraacetic acid, and 5 mM dithiothreitol. Following centrifugation (20 mins, 12 000 g), 2 mL of extract was desalted on 5 mL sephadex G25 (medium bead) columns and the enzyme measurements made on desalted extract. Ascorbate peroxidase was measured as described by Wang, Jaio & Fuast (1991) and glutathione reductase was measured as described by Sgherri et al. (1994b). Enzyme activities were calculated per mg protein [measured using the Bradford (1979) assay]. Due to large discrepancies among species, for comparative purposes the enzyme activities are expressed as a percentage of that typical of control, hydrated (pretreated) leaves. Ultrastructural studies Mesophyll leaf segments (approximately 5 mm2) were processed for transmission electron microscopy (TEM) using the method previously reported for these species (Farrant et al. 1999; Farrant 2000). Tissues were sectioned using a Reichert Ultracut-S microtome (Vienna, Austria), stained with uranyl acetate and lead citrate (Reynolds 1963) and viewed with a Jeol CX TEM (Jeol, Tokyo, Japan). RESULTS Table 1 gives the final water content (RWC) of leaves from plants subjected to the various treatments and summarizes the ability of the three species used in this study to recover and resume growth following the various treatments imposed. All treatments resulted in drying of the respective species to similar water contents, these being £ 5% RWC. Craterostigma wilmsii survived only if dried normally (leaves unrestrained) in the light. Plants dried in the light with leaves restrained died, although any leaves not restrained folded normally and survived. Myrothamnus flabellifolius survived drying in the light, regardless of whether leaves were restrained or not. Neither of these two species survived if dried in the dark, regardless of whether leaves were restrained or not. Xerophyta humils survived drying both in the light and dark. Electrolyte leakage Electrolyte leakage, which gives an indication of the degree of integrity of membranes, from leaves of variously treated plants is given in Fig. 1. There was no difference in extent of leakage, compared to hydrated material, in leaves of C. wilmsii which had been dried unrestrained in the light, nor upon their rehydration, suggesting little membrane damage occurred during these treatments (Fig. 1a). However, when leaves were restrained during drying in the light there was Table 1. Survival of Craterostigma wilmsii, Myrothanmus flabellifolius and Xerophyta humilis when dried normally or with leaves restrained under light or dark conditions Light unrestrained Light restrained Dark unrestrained Dark restrained Species Survival RWC Survival RWC Survival RWC Suvival RWC C. wilmsii M. flabellifolius X. humilis Yes Yes Yes 4.1% (±1.2) 3.0% (±0.95) 5% (±1.0) No Yes 4.0% (±0.99) 3.0% (±0.99) No No Yes 4.0% (±0.96) 3.1% (±1.0) 5% (±0.99) No No 4.0% (±1.1) 3.0% (±0.95) The final air-dry water contents [expressed as relative water content (RWC)] of leaves from plants at each treatment are given. Standard deviations are given in parentheses. © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 1278 J. M. Farrant et al. considerable increase in leakage in leaves from both dried and rehydrated plants, suggesting that this treatment caused a change in membrane configuration, probably damage, which was not repaired upon rehydration. Leaves from plants dried in the dark, whether restrained or not, also had increased electrolyte leakage in the dry and rehydrated state. The extent of leakage from plants dried in the dark was similar regardless of whether leaves were restrained or not, suggesting that lack of light rather than the restraining treatment, was the primary cause of membrane damage in these plants. Leaves of M. flabellifolius dried in the light, whether restrained or not, had a similar extent of electrolyte leakage compared to leaves from hydrated and rehydrated plants (Fig. 1b). Thus prevention of leaf folding during drying in the light did not appear to cause membrane damage as was the case for C. wilmsii. However, as for C. wilmsii, leaves from plants dried in the dark, both restrained and unrestrained, had increased electrolyte leakage compared with those from hydrated plants and this was exacerbated in leaves from rehydrated plants. Drying in the dark thus also appeared to have allowed some membrane damage in this species, which was not repaired upon rehydration. There was no change in electrolyte leakage in leaves of X. humilis dried in the light or in the dark, nor upon rehydration of the plants. Unlike the previous two species, drying in the dark did not appear to cause membrane damage in this species. Pigments Figure 1. Electrolyte leakage, expressed as a percentage of maximum possible leakage, in leaves of C. wilmsii (a), M. flabellifolius (b) and X. humilis (c). Black bars, control hydrated leaves; white bars, dry leaves; hatched bars, rehydrated leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark restrained. Error bars indicate standard deviation (n = 5). The exposure of chlorophyll to light during dehydration can lead to formation of free radicals which can be extremely damaging to tissues. In C. wilmsii, the chlorophyll content of leaves was reduced during drying in all treatments except those plants with leaves dried restrained in the dark (Fig. 2a). Restrained leaves dried in the light lost somewhat more chlorophyll than unrestrained ones. Only plants dried normally in the light were able to resynthesize chlorophyll during rehydration. In all other treatments, further chlorophyll breakdown occurred after prolonged rehydration (not shown) as the plants senesced. In M. flabellifolius, some loss of chlorophyll also occurred during drying in all treatments, but, as for C. wilmsii, those dried in the light with leaves restrained had greatest loss of chlorophyll (Fig. 2d). Upon rehydration, plants dried in the light were able to resynthesize chlorophyll. Plants dried in the dark lost chlorophyll as they senesced. Xerophyta humilis leaves Figure 2. Pigment contents of leaves of C. wilmsii (a, b, c), M. flabellifolius (d, e, f) and X. humilis (g, h, i). Black bars, control hydrated leaves; white bars, dry leaves; hatched bars, rehydrated leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark restrained. Chlorophyll contents are shown in the left-hand panel (a, d, g); carotenoid contents in the middle panel (b, e. h) and anthocyanin content in the right-hand panel (c, f, i). Error bars indicate standard deviation (n = 3). © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 Desiccation and light stress 1279 © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 1280 J. M. Farrant et al. lost virtually all chlorophyll when dried in the light (as is normal, since the plant is poikilochlorophyllous), but only half when dried in the dark (Fig. 2g). Chlorophyll was completely recovered during rehydration in both treatments. Carotenoids can protect against light-induced free radical formation (Larson 1988; Smirnoff 1993). The carotenoid content of leaves of C. wilmisii followed the same trend as that of chlorophyll, with some loss occurring during drying in all treatments except plants with restrained leaves dried in the dark, and only plants dried normally in the light being able to maintain carotenoids at original levels when rehydrated (Fig. 2b). Similarly, the carotenoid content of M. flabellifolius followed the same trends as occurred in the chlorophyll content in this species (Fig. 2e). Xerophyta humilis leaves lost 50% of their carotenoids in the light but only 10% in the dark. These pigments were recovered upon rehydration in both treatments (Fig. 2h). Anthocyanins are believed to mask chlorophyll from light, so preventing free radical formation, and also act as antioxidants (Larson 1988). These pigments were accumulated during drying in leaves of C. wilmsii regardless of the light regime given (Fig. 2c). Although the greatest levels accummulated in restrained leaves dried in the light, the fact that these pigments accummulated in plants dried in the dark suggests that it is in response to drying, rather than light. Anthocyanin content declined during rehydration in all treatments, a normal response in healthy plants (Farrant 2000) but probably due to senescence in those treatments (LR, DU, DR) which killed the plants. In M. flabellifolius anthocyanin content increased only in leaves of plants dried in the light, with slightly higher levels being recorded in those with unrestrained leaves (Fig. 2f). The amount of anthocyanin accummulated in this species was greater than in C. wilmsii and it would appear that in M. flabellifolius (compare Fig. 2c & f), anthocyanin accummulation might well be a light-mediated response. Levels dropped upon rehydration in all treatments. In X. humilis, low levels (compared with the other species) of anthocyanins accummulated in leaves dried in the light, although leaves dried in the dark also accummulated some anthocyanin. Like the other two species, anthocyanin content declined again upon rehydration (Fig. 2i). Antioxidants Changes in activity of the antioxidant enzymes ascorbate peroxidase (AP) and glutathione reductase (GR), are presented in Fig. 3 as a percentage of their activity in control, hydrated leaf tissues (where control = 100%). In C. wilmsii, AP activity increased during drying by 80–100% in all treat- Figure 3. Activity of the antioxidant enzymes ascorbate peroxidase (AP) and glutathione reductase (GR) as a percentage of activity in control, hydrated leaves, of C. wilmsii (a, b), M. flabellifolius (c, d) and X. humilis (e, f). White bars, dry leaves; hatched bars, rehydrated leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark restrained. Error bars indicate standard deviation (n = 5). © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 Desiccation and light stress 1281 ments, but only plants dried normally in the light reduced activity to that of control levels upon rehydration. Activity remained high (with considerable variation in the data) in restrained leaves from plants dried in the light and in leaves from dark-treated plants. GR activity increased markedly (to 275% of control) during drying in leaves of plants dried normally in the light, although some increase (< 40%) occurred in other treatments. On rehydration, the activity of this enzyme dropped to control levels (light unrestrained leaves) or below (Fig. 3b). Although protection afforded by AP was similar among treatments, GR activity was reduced in plants given treatments which killed them (LR, DR, DU). The lack of sufficient antioxidant protection would have allowed free radical damage to accrue, probably contributing to the death of plants from those treatments. In M. flabellifolius AP activity increased only in plants dried in the light, with significantly greater increases in activity occurring in leaves which had been restrained. On rehydration, activity remained high for longer periods in plants dried normally in the light; activity in restrained leaves dried in the light returned rapidly to control levels (Fig. 3c). Similar trends were observed for GR activity (Fig. 3d), although plants dried normally in light had higher GR activity on drying than those with restrained leaves. Plants dried in the dark had no measurable activity on drying nor on rehydration. The lack of increased antioxidant activity in plants which died (DU and DR treatments) could have allowed for accrual of free radical-associated damage, which in turn contributed to their death. AP activity increased markedly in leaves of X. humilis dried in the light, but there was no significant change in activity in leaves of plants dried in the dark (Fig. 3e). The activity of this enzyme returned to control levels during rehydration of plants dried in the light and became elevated during rehydration of plants dried in the dark. This pattern of activity may suggest that light is required for activation of the enzyme (which could occur during rehydration of plants dried in the dark, since rehydration occurred in the light). Such elevated activity during rehydration might have been required to ameliorate against ROS formation due to the presence of chlorophyll (Fig. 2c) and partially intact thylakoid membranes (Fig. 7b) present in tissues dried in the dark. GR activity was elevated during drying of plants dried both in the light and dark, although considerably more so in plants dried in the dark. Activity declined to control levels during rehydration in both treatments (Fig. 3e). Sucrose content Non-reducing sugars, sucrose in particular (Ingram et al. 1997; Ghasempour et al. 1998; Oliver, Hincha & Crowe 2001; Whittaker et al. 2001), are thought to protect the subcellular milieu against desiccation damage in a number of different ways. As some treatments involved dehydration in the dark, photosynthesis and thus sucrose accummulation might have been compromised, and so sucrose content during drying and rehydration was determined (Fig. 4). The Figure 4. Sucrose content of leaves of C. wilmsii (a), M. flabellifolius (b) and X. humilis (c). Black bars, control hydrated leaves; white bars, dry leaves; hatched bars, rehydrated leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark restrained. Error bars indicate standard deviation. (n = 3). sucrose content of leaves of C. wilmsii dried in the light increased markedly, regardless of whether leaves were restrained or not (Fig. 4a) to levels previously reported for this species (Cooper & Farrant 2002). Leaves dried in the © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 1282 J. M. Farrant et al. dark accummulated only half the amount of sucrose accummulated by those plants dried in the light. This indicates that photosynthesis, and/or a light-regulated switch for genes necessary for sucrose storage upon drying, is required for maximual accumulation of this protectant. On rehydration, sucrose declined in plants dried normally in the light, but not in those with restrained leaves dried in the light, nor unrestrained dried in the dark. This suggests that enzymes for utilization of sucrose might have been compromised by these dyring treatments. Plants with restrained leaves in the dark lost all sucrose, possibly being leached from the damaged tissues. In M. flabellifolius, sucrose accummulated during drying in leaves from all treatments (Fig. 4b) indicating that neither restraining, nor lack of light, interfered with accumulation of this protectant sugar. Upon rehydration, sucrose declined only in plants dried in the light indicating its utilization in metabolism in these plants but not in those dried in the dark. Sucrose content also increased in X. humilis plants regardless of whether they were dried in the light or dark and declined upon rehydration in plants dried in the light but not in those dried in the dark (Fig. 4c). Thus like M. flabellifolius, this species did not need to photosynthesize to produce adequate sucrose levels. It is unlikely that lack of mobilization of sucrose during rehydration of dark-dried plants was due to damage of enzymes required for this process. The plants had retained much of their chlorophyll and thus photosynthesis could be initiated almost immediately on full rehydration, supplementing the sucrose content and precluding measurable decline in this sugar. However, it is also possible that the enzymes for sucrose a c b d metabolism required de novo transcription and/or translation which delayed sucrose mobilization upon rehydration. Ultrastructural status Mesophyll cells from leaves of C. wilmsii dried unrestrained in the light had subcellular organization typical of that reported previously (Farrant et al. 1999; Farrant 2000) and the data are not shown. Cells from dry leaves had folded walls and the plasmalemma was closely adhered to them. Thylakoid membranes were intact, albeit displaced to one side of the chloroplast. There were no obvious signs of damage in the dried or rehydrated state. Mesophyll cells from restrained leaves dried in the light and those dried in the dark (whether restrained or not) had signs of subcellular damage (Fig. 5a & b). Although wall folding was evident in all treatments (arrowed in Fig. 5a) drying in the dark resulted in considerable plasmalemma withdrawal (more than what could be ascribed to conventional fixation, if one compares tissue dehydrated unrestrained in the light) and loss of integrity (arrowed in Fig. 5b). Chloroplasts from tissues dried in the light did not appear damaged and had well-organized thylakoid membranes, but numerous plastoglobuli were present in some (Fig. 5a). Chloroplasts from plants dried in the dark had blistered thylakoid membranes (Fig. 5b). All damage was considerably exacerbated when these plants were rehydrated (Fig. 5c & d). Complete loss of plasmalemma and organellar integrity occurred. Mesophyll cells from leaves of M. flabellifolius dried in the light showed no sign of damage, regardless of whether the leaves were restrained or not and only those from Figure 5. Subcellular organization of mesophyll cells of Craterostigma wilmsii dehydrated (a) in the light (LR), and (b) in the dark (DR); and rehydrated after drying with leaves restrained (c) in the light and (d) in the dark. Note the plasmalemma withdrawal and folded walls (arrowed), small vacuoles (v), plastoglobuli (p) in chloroplasts (c) in (a). In (b), vacuoles were absent, plasmalemma withdrawal and rupture (arrowed) was evident, thylakoids were blistered. Rehydrated tissue (c & d) showed total disruption of plasmalemma and general cytoplasmic dissolution. © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 Desiccation and light stress 1283 a b c d restrained tissues are shown. There was no plasmalemma withdrawal from the cell wall and vacuoles containing some relatively electron dense material were evident (Fig. 6a) as previously described for tissues dried under normal conditions (Farrant 2000; Farrant & Kruger 2001). Thylakoid membranes were blistered, but this too is normal for this species (Koonjul et al. 2000). On rehydration subcellular organization was typical of hydrated unrestrained leaves (Sherwin & Farrant 1996; Farrant & Kruger 2001). A single large central vacuole had reformed in each cell and thylakoid membranes had become restacked in the typical ‘staircase’ formation, with plastoglobuli present (Fig. 6b). Dehydration in the dark resulted in no obvious subcellular damage in neither restrained nor unrestrained leaves (Fig. 6c). However, upon rehydration there was general dissolution of subcellular membranes with few organelles remaining intact (Fig. 6d). Leaf tissues of X. humilis dried in the light had no subcellular damage and were typical of that previously reported (Farrant et al. 1999; Farrant 2000). Several smaller vacuoles (compared to the one central vacuole, typical of hydrated material) were evident in the cytoplasm and thylakoid membranes were no longer present within chloroplasts (Fig. 7a). When dried in the dark, thylakoid membranes were only partially dissembled (Fig. 7b) but all other organelles were intact and resembled those of plants dried in the light. Upon rehydration, subcellular orgaization was fully recovered in both treatments (Fig. 7c). DISCUSSION It is well documented that light energy harnessed by chlorophyll, which cannot be dissipated via photosynthesis as Figure 6. Subcellular organization of mesophyll cells of Myrothamnus flabellifolius on dehydration (a) and then rehydration (b) of leaves restrained and dried in the light. (c) Mesophyll cells of leaves restrained dried in the dark and (d) following rehydration. Note blistered thylakoid membranes in chloroplasts [c] and vacuole [v] with relatively electron dense material in (a). Thylakoids had reformed and plastoglobuli were evident in chloroplasts in (b). Numerous small vacuoles and chloroplasts with blistered thylakoids were evident in (c) and plasmalemma damage and general dissolution of cytoplasm was evident in (d). occurs under water-limiting conditions, can lead to the formation of oxygen free radicals (Halliwell 1987; Kaiser 1987; Larson 1988; Smirnoff 1993; Navari-Izzo et al. 1997b). We have previously proposed that the homoiochlorophyllous resurrection plants C. wilmsii and M. flabellifolius are able to minimize such free radical formation, in part, by leaf folding which minimizes light–chlorophyll interaction in inner leaves of the former and adaxial surfaces of the latter (Sherwin & Farrant 1998; Farrant 2000). In the present study we attempted to test this suggestion by preventing leaf folding during drying of these species. Such treatment did indeed lead to loss of viability in restrained leaves of C. wilmsii but not in the case of M. flabellifolius. Although not unequivocal, it is likely that light stress during dehydration contributed significantly to the loss of viability in restrained leaves of C. wilmsii. All leaves that were prevented from folding, either the outermost layer or any of the inner leaves, died when dried in the light. Restrained leaves dried in the dark did not suffer any more (or different) damage to unrestrained leaves, so it was unlikely to be the restaining per se that caused viability loss. Evidence of light stress in restrained leaves are the increased number of plastoglobuli in chloroplasts (not usually present in chloroplasts from dry tissue in this species) (Fig. 5a), reduced chlorophyll and elevated anthocyanin content of restrained leaves (Fig. 2c). Interestingly, restrained leaves had either similar, or elevated levels of putative protectants [anthocyanins (Fig. 2c), sucrose (Fig. 4) and AP activity (Fig. 3a)] than in plants dried normally in the light. Presumably this protection was insufficient to prevent lethal damage [measured here as increased electrolyte leakage (Fig. 1a) and loss of subcellular compartmentation (Fig. 5a & c)] and loss of viability. We have © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 1284 J. M. Farrant et al. a b Figure 7. Subcellular organization of mesophyll cells of Xerophyta humilis dried in the light (a), in the dark (c) and following rehydration after drying in the dark (c). Note dismantled thylakoid membranes and plastoglobuli [p] in chloroplasts [c] and small vacuoles [v] in (a). In (b), thylakoid membranes had not totally dismantled. On rehydration (c) thylakoids reformed and starch [s] was present. c shown using metabolic inhibitors of transcription and translation that this species relies predominantly on establishing protection during drying, rather than repair on rehydration (Cooper & Farrant 2002). Although repair can occur, it was presumably also insufficient to prevent loss of viability here. Two explainations can be advanced to account for loss of viability in restrained leaves: 1 Protective mechanisms are only up-regulated in abaxial cells which normally get exposed to light during drying, and thus were not (sufficiently) present in adaxial tissues to adequately protect them from light stress in restrained leaves. Since whole leaves were sampled for the biochemical studies, we cannot differentiate between the protection content of abaxial versus adaxial surfaces. In partial support of this theory, abaxial surfaces of restrained leaves (light- and dark-dried) were always noticeably more purple, reflecting increased anthocyanin content. 2 Outer leaves suffer light stress during normal drying, but are ‘sacrificed’ in order to protect the inner leaves. In C. plantigineum, it is reported that outer leaves rarely resurrect and die back after each cycle of drying and rehydration (Norwood et al. 1999, 2000). Usually, in C. wilmsii, the outer leaves will resurrect for two to three cycles of drying and rehydration before dying (personal observation), suggesting some degree of protection and/ or ability to repair damage in these leaves. We propose that a combination of above explanations is true. That is, abaxial surfaces have a greater ability to protect and repair against light stress, but these leaves become increasingly damaged with repeated cycles of drying and rehydration and are ultimately lost. The advantage of this strategy of leaf folding is that chloroplast structure and chlorophyll of inner, shaded leaves can be retained (Sherwin & Farrant 1996, 1998; Farrant 2000) and photosynthesis can start quickly upon rehydration in inner leaves and recovery of the plant is rapid (Sherwin & Farrant 1996). In the poikilochlorophyllous resurrection plants such as X. humilis, where thylakoids are dismantled and chloropyll is broken down (Fig. 2g), recovery is far slower (Sherwin & Farrant 1998; Tuba et al. 1998) but all leaves recover. In M. flabellifolius, restraining does not cause subcellular damage and loss of viability of leaves dried in the light. In this species, leaf folding normally shades only the adaxial surface of each leaf; our experiments served only to swop the surface exposed to light during drying. Thus in this species it appears that both ad- and abaxial cells are able to protect against light stress. Furthermore, abaxial surfaces, at least, must be able to undergo repeated cycles of drying and rehydration without accrual of damage such that leaves are lost. Indeed the degree of protection in leaves of this species is considerable; AP and GR activity (Fig. 3c & d), and anthocyanin levels (Fig. 2f) are far higher in M. flabellifolius than in C. wilmsii or X. humilis. In addition, thylakoid membranes become unstacked during drying (Fig. 6a) a feature suggested to put a stasis on photosynthesis and minimize ROS formation (Koonjul et al. 2000; Farrant 2000). Although this strategy slows the rate of recovery of photosynthesis during rehydration (Sherwin & Farrant 1996; Farrant & Kruger 2001) relative to C. wilmsii, the trade-off is in retention of all leaves during drying and rehydration, at least for more cycles of drying and rehydration than observed for C. wilmsii. It is also possible that this © 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286 Desiccation and light stress 1285 species has better ability to repair light-induced damage on rehydration (Farrant & Kruger 2001). The intriguing aspect of this work is that the homoiochlorophyllous species did not survive drying in the dark, whereas the poikilochlorophyllous X. humilis did. The plants were not held in the dark for long periods, so it is unlikely that death was due to ‘starvation’ [indeed, M. flabellifolius synthesized as much sucrose in the dark as plants dried in the light (Fig. 4b)]. Many genes, nuclear and chloroplastic, are light regulated (Terzaghi & Cashmore 1995) and it is possible that at least some of the genes responsible for laying down of protection against desiccation in homoichlorophyllous species examined here, require light, in combination with water deficit, for their transcription/ regulation. Indeed Early Light Inducable Genes (ELIP) have been shown to be up-regulated in Craterostigma plantigineum (Alamillo & Bartels 2001; Phillips et al. 2002) and in the resurrection moss Tortula ruralis (Zeng, Chen & Woods 2002), and are believed to play a role in protection against photoinhibition during early stages of dehydration. Since the poikilochlorophyllous X. humilis survived drying in the dark it is possible that light-induced regulation of protection mechanisms does not occur in this species. Furthermore, since the major difference between homoiochlorphylly and poikilochlorophylly is the maintenance of chlorophyll and intact photosystems during drying in the former, it is tempting to speculate that these might be important in signal transduction of desiccation-related protection mechanisms in the homoiochlorophyllous types. Our data suggests that light is necessary for increased activity of antioxidant enzymes and anthocyanin accumulation in M. flabellifolius but not in C. wilmsii. Although it is not possible to distinguish here whether light does indeed act as a signal for up-regulation of these protectants, we propose that their increased levels in leaves of M. flabellifolius dried in the light is necessary because this species is afforded less protection against light–chlorophyll interaction by leaf folding than is C. wilmsii. In the latter, all the inner leaves (> 90% of the plant) are shaded from light, with only the abaxial surfaces of outer leaves requiring antioxidant and anthocyanin protection and a light-driven regulation of such systems may not be as vital. ACKNOWLEDGMENTS The authors thank John and Sandy Burrows for the collection of C. wilmsii and Borakalalo National Park for allowing collection of X. humilis. 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