Annals of Botany 88: 1093-1100, 2001 doi:10.1006/anbo.2001.1552, available online at http://www.idealibrary.com on IDEK1 ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum NOSISA MAYABAf, RICHARD P. BECKETT*t, ZSOLT CSINTALANJ and ZOLTAN TUBA} ^School of Botany and Zoology, University of Natal, Pietermaritzburg, Private Bag X01, Scottsville 3209, South Africa and \Department of Botany and Plant Physiology, Faculty of Agricultural and Environmental Sciences, Sr. Istvan University, H-2103, Godollo, Pater K. u. 1, Hungary Received: 5 February 2001 Returned for revision: 30 April 2001 Accepted: 5 September 2001 Key words: Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence. during desiccation, especially if plants are illuminated (Seel etai, 1992). Most bryophytes are desiccation-tolerant organisms, and Bryophytes appear to survive desiccation through a can survive in the air-dried state for long or short periods complex interplay of many mechanisms rather than one even at relative water contents (RWC) below 10%, a state simple adaptive feature (Oliver, 1996; Oliver and Bewley, that would be lethal to all non-desiccation tolerant plants 1997; Oliver and Wood, 1997; Oliver et al., 1997, 1998). For (Gaff, 1997; Kranner and Lutzoni, 1999). Although example, some species have been shown to possess dehydrin desiccated bryophytes appear completely dry, they can proteins that may act as surfactants, inhibiting the revive and rapidly show normal physiological characteristics coagulation of a range of macromolecules (Hellwege el al., during rehydration (Tuba et ai, 1996). Desiccation is 1994, 1996). Removal of ROS produced during desiccation harmful to plants for many reasons (Oliver et ai, 1998): by antioxidants is also important. Antioxidants include the cytoskeleton may be damaged as a result of the large molecules such as the tri-peptide glutathione, ascorbic acid changes in cell volume that accompany desiccation; water or tocopherols, and the enzymes superoxide dismutase, removal can lead to membrane damage, increased ionic catalase and peroxidases (for a review see Kranner and strength and a change in pH, causing crystallization of Lutzoni, 1999). Furthermore, non-photochemical quenchsolutes and protein denaturation; and water deficit also ing (NPQ) can reduce ROS production by dissipating excess induces the formation of reactive oxygen species (ROS) such energy using the xanthophyll cycle (Gilmore, 1997). The as singlet oxygen ('O2), the hydroxyl radical (OH), the xanthophyll cycle involves stepwise removal (de-epoxidasuperoxide anion radical (O2~) and hydrogen peroxide tion) of two oxygen functions (the epoxy groups) in (H2O2) (Smirnoff, 1993; Minibayers and Beckett, 2001). violaxanthin to form zeaxanthin, and requires light, ROS can react with many classes of biomolecules and thus ascorbate and reducing conditions, the latter probably a can damage almost all cellular compartments. Although consequence of cyclic electron flow (Heber et al., 2000). reactive oxygen species are produced during normal Desiccation-tolerant bryophytes appear to possess high metabolism, especially in chloroplasts and mitochondria NPQ, particularly at low water contents (Deltoro et al., (Halliwell, 1987), desiccation greatly enhances their pro- 1998a, b; Csintalan et al., 1999). In the resurrection angioduction. Chlorophyll breakdown can also occur in mosses sperm Craterostigma, desiccation tolerance appears to depend upon an increase in sucrose during desiccation, * For correspondence. Fax + 27 33 2605 105, e-mail beckett&nuac. presumably to promote vitrification of the cytoplasm and protect membranes (Ingram and Bartels, 1996; Scott, 2000). za INTRODUCTION 0305-7364/01/121093 + 08 S35.00/00 % 2001 Annals of Botany Company Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 The effect of pretreatment with abscisic acid (ABA) on the physiology of the moss Atrichum androgynum during a desiccation—rehydration cycle was examined. During rehydration following desiccation for 16 h, net CO 2 fixation recovered much more slowly than photosystem II (PSII) activity, conditions conducive to the formation of reactive oxygen species (ROS) in the photosynthetic apparatus. Pretreatment with ABA increased the rate of recovery of photosynthesis and PSII activity, and also doubled non-photochemical quenching (NPQ). Increased NPQ activity will reduce ROS formation, and may explain in part how ABA hardens the moss to desiccation. In ABA-pretreated, but not untreated mosses, desiccation significantly increased the concentration of soluble sugars. Sugar accumulation may promote vitrification of the cytoplasm and protect membranes during desiccation. Starch concentrations in freshly collected A. androgynum were only approx. 40 mg g" 1 dry mass; they rose slightly during desiccation but were only slightly affected by ABA pretreatment. ABA did not reduce chlorophyll breakdown during desiccation. © 2001 Annals of Botany Company 1094 Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum membranes. In addition, ABA treatment could preserve chlorophylls during desiccation. The overall aim of these experiments was to investigate further the role of ABA in desiccation tolerance in A. androgynum. M A T E R I A L S A N D METHODS Plant material Atrichum androgynum (C. Mull.) A. Jaeger was collected during the moist summer months from the understorey of the Doreen Clarke Nature Reserve, Hilton, KwaZulu Natal Province, Republic of South Africa (24=39'S and 30=17'E). This nature reserve is a small pocket of Afromontane forest in the mist belt region of KwaZulu Natal. Once collected, material was stored on wet filter paper for 2 d at 20 °C and a light intensity of 75 umol photons m~2 s"1 under continuous fluorescent light. Apical segments 2 cm long were then cut, and for all experiments except those involving carbohydrate and chlorophyll analyses, material was divided into five replicates of ten segments. Each replicate typically had a fresh mass of 160 mg and a dry mass of approx. 50 mg. For carbohydrate analyses, 500 mg fresh weight of material was used, and for chlorophyll analyses 200 mg fresh weight was used. Desiccation and rehydration cycle Abscisic acid (+ cis-trans, Sigma) was dissolved in a drop of 1 M NaOH, and the pH of the resulting solution adjusted to 5-6 with HC1. Mosses were pretreated with 100 UM ABA or distilled water for 1 h and stored hydrated for 3 d at 20 °C and 75 umol photons m~2 s~'. They were then desiccated for 16 h by placing the stem segments in 2 x 5 cm specimen bottles in a desiccator over silica gel at 20 °C and a light intensity of 75 umol photons m~2 s~' under continuous fluorescent light [see Beckett (1999) for a graph of RWC as a function of time]. The mosses were rehydrated suddenly by addition of 10 ml distilled water. Rehydration was probably complete within 2 min. In preliminary experiments, mosses were desiccated for 8, 16, 24 and 30 h to determine the most appropriate duration for desiccation stress. Results indicated that desiccation for 16 h significantly inhibited photosynthesis during the first few hours of rehydration, but that after 8 h of rehydration net photosynthesis had partially recovered (Fig. IB). Mosses were desiccated for 16 h in all subsequent experiments. During desiccation, the RWC of plants dropped to 015 after 8 h and to 004 after 16 h. Photosynthesis, respiration, chlorophyll fluorescence parameters, and the concentrations of soluble sugars, starch and chlorophylls were measured in freshly collected material after pretreatment with ABA or water for 3 d, then at intervals during the 8 h following rehydration. Plants were normally rehydrated under dim laboratory lighting (approx. 5 umol photons m~2 s~'), except in the experiment measuring respiration when they were rehydrated in complete darkness. In addition, for measurement of net photosynthesis, mosses were exposed to 150 umol photons m~2 s~' for the 10 min during which readings of photosynthesis were taken. Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 However, the preliminary studies of Smirnoff (1992) found no increase in sugar concentration during desiccation in a range of mosses. Beckett and co-workers have used the moss Atrichum androgynum as a model system for studying desiccation tolerance in mosses. This species, which is common in the understorey of Afromontane forests, combines the upright habit of Polytrichum with the fragile textured leaves of Mnium. In initial experiments, Beckett and Hoddinott (1997) measured desiccation tolerance using a simple ion leakage assay which involves measuring K.+ loss 30 min after rehydration following desiccation of fully hydrated material over silica gel. Large seasonal changes in K + leakage occur in A. androgynum following desiccation. Mosses lose much less K + during the dry winter months than in the moist summer months, indicating the presence of inducible tolerance mechanisms that reduce desiccationinduced damage. Later studies showed that this species could be hardened to desiccation stress under controlled conditions. Reducing the RWC to approx. 0-6 for 3 d increases tolerance, even if plants are then stored fully hydrated for 24 h before assaying for desiccation tolerance (Beckett, 1999). Furthermore, exogenous applications of abscisic acid (ABA) followed by storage for 3 d can fully substitute for partial dehydration. Presumably, in the field, partial dehydration will often precede a more severe desiccation stress, inducing ABA synthesis which in turn activates signal transduction pathways that increase desiccation tolerance. These results, and other limited data available in the literature, strongly suggest that ABA plays an important role in the induction of genes that harden A. androgynum, and at least some other bryophytes, to desiccation stress (Hartung et al., 1998). Beckett el al. (2000) used chlorophyll fluorescence to show that in the closely related species A. undulatum, ABA pretreatment speeds up the recovery of photosystem II (PSII) during rehydration following desiccation, and also increases NPQ. If desiccation reduces Calvin cycle activity more than membrane-bound electron transport, excess excitation energy can pass to oxygen from photo-excited chlorophyll pigments, forming singlet oxygen, while superoxide and hydrogen peroxide are produced at PSII (McKersie and Lesham, 1994). Under these conditions enhanced NPQ may reduce potentially harmful ROS formation, and may explain how ABA increases the desiccation tolerance of PSII and reduces ion leakage. The first aim of the work presented here was to compare time courses of the recovery of respiration and carbon fixation with PSII activity during rehydration following desiccation. Results obtained confirmed that PSII activity recovers much faster than C fixation, suggesting that NPQ acts as a 'safety valve', reducing ROS production when Calvin cycle activity is impaired. We also studied the effects of ABA pretreatment on the concentrations of carbohydrates and chlorophylls in A. androgynum during desiccation and rehydration. We hypothesized that in addition to its effects on NPQ, ABA treatment could increase the pool of soluble sugars available during desiccation. As discussed above, sugar accumulation will help vitrify the cytoplasm or preserve Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum 1095 8 "3 e 0 3.5 cd 3.0 100 200 300 Light intensity (umol photons nv2 400 A = start of experiment B = after 3 d A = start of experiment B = after 3 d O O "o a o J 0 <A • ABA-treated o untreated | I 2 Time (h) F I G . 1. A, The effect of light intensity on photosynthesis in the moss A. androgynum. Points represent fitted values with 95 % confidence limits calculated using the 'Spline' program of Hunt and Parsons (1974). B, The effect of desiccation for a range of times on photosynthesis during rehydration in the moss A. androgynum. In this and subsequent figures, points represents the means, and error bars the standard deviations. Overlapping error bars have been removed. The effect of treatment with distilled water or 100 UM ABA for 1 h followed by storage for 3 d and then desiccation for 16 h on respiration (C) and net photosynthesis (D) during rehydration in the moss A. androgynum. In D, an asterisk indicates a significant difference between ABA-pretreated and untreated mosses (Student's /-test, P < 005). Measurement of photosynthesis and respiration recorded. The actinic light was then switched on, the initial peak (Fp) noted, and F (steady-state fluorescence during Net photosynthesis (A) and respiration were measured at illumination), Fm (maximum fluorescence following 25 °C and a relative humidity of 50 % using an Analytical illumination), and (with the actinic light switched off) Fo Development Corporation (ADC) Mark III portable infra(minimum fluorescence following illumination) were red gas analyser (IRGA) with a barrel-shaped Parkinson recorded after 5 min, by which time a steady state had leaf chamber, modified with a water-cooled jacket. The flow been reached. Calculation of fluorescence parameters and 1 rate through the leaf chamber was 120 ml min" . In preconventions for symbols follows Schreiber and Bilger (1993) liminary experiments, photosynthesis was found to saturate and Schreiber et al. (1995); the Stern-Volmer quotient NPQ 2 at 150 umol photons m~ s~' (Fig. 1A). Equilibrating fully was used as a measure of non-photochemical quenching. hydrated samples for 10 min was found to give steady-state Briefly, FJFm = (Fm- Fo)/Fm, d>PSII = (Fm-F)/Fm, rates of gas exchange without causing enough water loss to NPQ = FJFm-l; Fo quenching = {FO-FO)IFO , where Fv reduce photosynthesis. is the variable fluorescence after dark adaptation. Chlorophyllfluorescencemeasurements Chlorophyll fluorescence was measured using a Hansatech Instruments (King's Lynn, UK) FMS1 modulated fluorometer. Preliminary investigations showed that actinic light at 50 (imol photons m~2 s~' gave a good balance between photochemical and non-photochemical quenching. To take a measurement, each replicate was placed in the fluorometer, and Fo and Fm (respectively minimum and maximum fluorescence after dark adaptation) were Determination of soluble sugars and starch For soluble sugar analysis, 0-5 g (fresh mass) material was ground in liquid nitrogen and extracted three times with 5 ml 80 % ethanol at 75 =C for 5 min, centrifuging after each extraction. The supernatants were pooled and made up to 25 ml with distilled water. For starch analysis, 5 ml of distilled water was added to the remaining pellet, the tubes were heated at 100 °C for 5 min, then 5 ml of Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 T3 1096 Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum 60 % perchloric acid was added. Tubes were then centrifuged at 1500 g for 30 min, the supernatant decanted, and the pellet washed in 10 ml 15 % perchloric acid. Tubes were re-centrifuged, the supernatants pooled and then made up to 25 ml with distilled water. For both sugar and starch analyses, 0-4 ml 2 % phenol and 2 ml concentrated H2SO4 were added to 0-4 ml of extract and A490 measured. Determination of chlorophylls Chlorophylls were extracted by grinding 200 mg of plant material (fresh mass) in liquid nitrogen using a pre-chilled pestle and mortar, then extracting in 5 ml of 100 % acetone. Extracts were centrifuged at 1500 g and 4 °C for 6 min, and total chlorophylls (a and b) were measured spectrophotometrically using the equations of Lichtenthaler (1987). During storage for 3 d following treatment with distilled water or ABA, respiration increased slightly, declined to zero following desiccation, then increased rapidly during the first 20 min of rehydration (Fig. 1C). In untreated material, the rate of respiration increased from approx. 1 umol CO2 mg"' dry mass h~' before desiccation to approx. 3 umol CO2 mg" 1 dry mass h" 1 after 20 min rehydration, while in ABA-treated material respiration was significantly less at only approx. 1-7 umol CO2 mg" 1 dry mass h~' (Student's /-test, P < 001). After rehydration for 2 h, respiration in material from both treatments recovered to values similar to those prior to desiccation. The rate of net photosynthesis at the start of the experiment was approx. 4 umol CO2 mg" 1 dry mass h~' (Fig. ID). The rate declined slightly during storage following treatment with distilled water or ABA, and was slightly lower in ABA-treated material. Immediately following rehydration, a burst of CO2 was released. The rate of CO2 release was approx. 6 umol CO2 mg"' dry mass h~' in untreated material, but was significantly less (approx. 3 umol CO2 mg" 1 dry mass h~') in ABA-treated material (Student's /-test, P < 0001). Rates of CO 2 release were much faster than the respiratory rates of material rehydrated in the dark. Net photosynthesis recovered more slowly than respiration, but recovered faster in ABA-treated compared with untreated material. ABA-treated material required 2 h to reach positive carbon balance, while untreated material needed 8 h. Recovery was incomplete after 8 h; even ABAtreated material only achieved a net photosynthetic rate of approx. 1 umol CO, mg" 1 dry mass h" 1 . During storage following water or ABA pretreatment, minimum and maximum fluorescence (Fo and Fm) declined slightly, while other fluorescence parameters remained unchanged (Fig. 2A-F). Desiccation reduced all parameters except Fv/Fm sharply. During rehydration, Fo and Fm recovered quickly, with Fm recovering slightly faster in ABA-treated plants. Fv/Fm did not recover in untreated plants, but increased slightly in ABA-treated plants. The actual quantum yield of PSII (<j>PSII) initially declined further during rehydration, then recovered partially, but was consistently higher in ABA-treated plants. NPQ and Fo DISCUSSION Results presented here clearly indicate that PSII activity in the moss Atrichum androgynum recovers much more quickly than photosynthetic carbon fixation during rehydration following desiccation, supporting the limited data available for other mosses (Proctor and Smirnoff, 2000). As discussed in the Introduction, rapid recovery of PSII activity but slow recovery of carbon fixation can cause ROS production (McKersie and Lesham, 1994). Under these conditions, NPQ may considerably reduce the amount of oxidative damage. Pretreatment with ABA both increases NPQ (Fig. 2F) and improves the tolerance of Atrichum to desiccation-induced damage e.g. ion leakage (Beckett, 1999, 2001), and reductions in PSII activity (Beckett et al., 2000). It seems likely that in A. androgynum some of these beneficial effects are a consequence of increased NPQ activity. This is supported by the observation that FJFm declines if the moss Racomitrium is rehydrated with the xanthophyll cycle inhibitor dithiothreitol in the light, but not in the dark (Proctor and Smirnoff, 2000). The likely 'cost' of increased NPQ is reduced rates of photosynthesis at non-saturating light intensities (Niyogi et al., 1998; Niyogi, 2000). It is worth noting that in addition to these effects, ABA pretreatment also considerably reduces the respiratory burst of A. androgynum rehydrated in the dark (Fig. 1C). The observation that ABA increases desiccation tolerance even in the absence of light implies that ABA can improve tolerance by means other than through the beneficial effects of increased NPQ. However, ABA-induced increases in NPQ are likely to harden bryophytes to stresses that inhibit carbon fixation more than PSII activity. Few measurements of endogenous levels of ABA in bryophytes exist in the literature (for a review see Christianson, 2000). Slow drying increased the ABA concentration of the moss Funaria hygrometrica to 10 nmol g~' dry mass (Werner et al., 1991), which would correspond to approx. 5 UM in the cytoplasm immediately Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 RESULTS quenching recovered quickly, and were always much higher in ABA-treated plants. Initially, the concentration of soluble sugars was approx. 59 mg g"1 dry mass, and it increased slightly during storage (Fig. 3A). Following desiccation, the concentrations increased significantly to 106 mg g"1 dry mass in ABAtreated material (Student's r-test, P < 0-01), but to only 86 mg g~' dry mass in untreated material (ns, P > 01). After rehydration for 1 h, soluble sugar concentrations declined in both treatments, but tended to remain slightly higher in ABA-treated material, although the difference was not significant. Starch concentrations in freshly collected A. androgynum were only approx. 40 mg g~' dry mass; they rose slightly during desiccation but were only slightly affected by ABA pretreatment (Fig. 3B). Desiccation reduced the total concentration of chlorophylls a and b from approx. 6 to approx. 3-5 mg g~' dry mass, and concentrations had not recovered after 8 h rehydration (Fig. 3C). No differences existed in the responses of chlorophylls to desiccation in ABA- and non-ABA treated material. 1097 Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum f A 200 — 1 B \ B 150 100 50 h 1-Hl- | V -H^-l 1— i i 6 8 0.35 - 0.4 bo a £ 0.3 A B T Ji c \ O> 3 o- 0.2 0.1 0 1 „ I / T T T J / i ^ 1 JL—V'T / \ f1 u—JI 0 I I 2 I 4 6 8 Time (h) F I G . 2. The effect of treatment with distilled water or 100 \XM ABA followed by storage for 3 d then desiccation for 16 h on Fo (A), Fm (B), FJFm (C), Fo quenching (D), <t>PSII (E) and NPQ (F) in the moss A. androgynum. • , ABA-treated material; O, untreated material. after rehydration. In the present study, ABA was applied at 100 UM because Beckett (1999) found that this concentration significantly increased tolerance to desiccationinduced ion leakage during rehydration in A. androgynum. Low rates of ABA uptake or alternatively rapid metabolism of the ABA supplied (not measured) may explain why such high exogenous concentrations were needed to elicit a response. However, in order to confirm the role of ABA in the hardening of A. androgynum in field situations our future work will focus on endogenous ABA metabolism. ABA possibly increases NPQ activity by increasing the concentrations of xanthophyll cycle pigments (for a review see Gilmore, 1997). Recently, Bukhov et al. (200\a,b) showed that the main mechanism for dissipating light energy in the moss Rhytidiadelphus was zeaxanthindependent quenching in the antenna of photosystem II. Few studies exist on the effects of ABA on NPQ, although Ivanov et al. (1995) showed that ABA treatment increased both NPQ and the levels of xanthophyll cycle pigments in barley seedlings. Interestingly, Zorn et al. (2001) showed that slow desiccation and rehydration of the highly desiccation-tolerant lichen Ramalina maciformis causes conversion of zeaxanthin to violaxanthin and also de novo synthesis of violaxanthin. This presumably hardens the lichen to a subsequent, more severe stress (e.g. longer desiccation or more rapid rehydration). However, it is not known whether ABA is involved in desiccation tolerance in lichens (Dietz and Hartung, 1998, 1999). We intend to investigate the mechanism of increased NPQ by studying the effect of ABA on xanthophyll cycle pigments in Atrichum. Respiration rates measured during the drying and rehydration cycle also suggest that light-induced free radical Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 u.o 1098 Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum B dry 125 lo to £ A = start of experiment B = after 3 d A = start of experiment B = after 3 d 100 75 - A 50 bo en a 25 Sol 3 0 I I 0 2 Time (h) 6 P. o _o Tot 2 Flo. 3. The effect of treatment with distilled water or 100 UM ABA followed by storage for 3 d then desiccation for 16 h on the concentrations of soluble sugars (A), starch (B) and total chlorophylls (a + b) (C) during rehydration in the moss A. androgynum. An asterisk indicates a significant difference between ABA-pretreated and untreated mosses (Student's f-test, P < 0-05). • , ABA-treated material; O, untreated material. production is responsible for some of the harmful effects of desiccation. The respiratory burst was much larger when A. androgynum was rehydrated in the light than in the dark (Fig. \C and D). Compared with non-desiccated material, the increases in the rates of respiration during the early stages of rehydration in mosses not treated with ABA were 5 and 2 umol CO 2 g~' dry mass h~' in the light and the dark respectively. In ABA-treated material the increases were only 2 and 0-7 umol CO2 g~' dry mass h~' respectively. Other workers have also found that rehydration of mosses in the light is more harmful than in the dark. For example, Seel et al. (1992) found rehydration in the light increased chlorophyll breakdown in the moss Dicranum, strongly suggesting that light-generated free radicals are responsible for at least some of the harmful effects of desiccation on bryophytes. ABA treatment stimulates the accumulation of soluble sugars during desiccation from 59 to 106 mg g~' dry mass (Fig. 3A), while in untreated material the concentration only increased to 86 mg g~' dry mass. This experiment was repeated three times, and in every case the accumulation of soluble sugars during desiccation was always much higher in the ABA-treated moss. According to Santarius (1994), freshly collected material of the closely related species A. undulatum contains approx. 80 mg g~' dry mass of sugars, within the range of concentrations reported here for A. androgynum. Most of the sugar was sucrose, but plants also contained small quantities of fructose and glucose. As discussed in the Introduction, sugars can promote vitrification of the cytoplasm and can protect membranes (Scott, 2000). Increases in soluble sugars following desiccation in ABA-treated material were proportionally similar to those reported in the resurrection angiosperms Myrothamnus fabellifolia and Xerophyta viliosa, but were much less than those found in Cralerostigma plantagineum (for a review see Scott, 2000). Smirnoff (1992) found no increase in sugars during desiccation in a range of mosses, similar to the response of non-ABA treated material in the present study. However, Schwall et al. (1995) reported that ABA could induce sucrose accumulation in Craterostigma. It is possible that the ability to accumulate sugars during desiccation is only expressed in hardened mosses. The accumulation of soluble sugars may play a small, but possibly significant, role in the ABA-induced increase in desiccation tolerance in A. androgynum. Oliver et al. (1998) divide desiccation tolerant plants into: (1) those that survive if drying is slow enough to induce mechanisms that either protect the plants during desiccation or facilitate recovery during dehydration; and (2) those that tolerate rapid drying. These two strategies may be termed 'constitutive' or 'inducible', respectively. The advantage of inducible systems is that, unlike constitutive mechanisms, they do not divert energy away from growth and reproduction. The disadvantage is that a sudden, severe drought may not allow time for the induction of tolerance, and thus plants may not survive. Oliver et al. (2000) argue that Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 Ml Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum ACKNOWLEDGEMENTS We gratefully acknowledge the support of the South African-Hungarian Science and Technology Programme (DAK 9/98 Project), the University of Natal Research Fund, and the Research Development in Higher Education Budapest Fund (FKFP 0472/97). NM gratefully acknowledges the receipt of a National Research Foundation student bursary. LITERATURE CITED Beckett RP. 1999. Partial dehydration and ABA induce tolerance to desiccation-induced ion leakage in the moss Atrichum androgynum. South African Journal of Botany 65: 212-217. Beckett RP. 2001. ABA-induced tolerance to desiccation-induced ion leakage in the moss Atrichum androgynum. Plant Growth Regulation 35 (in press). Beckett RP, Hoddinott N. 1997. Seasonal variations in tolerance to ion leakage following desiccation in the moss Atrichum androgynum from a KwaZulu Natal Afromontane Forest. South African Journal of Botany 63: 276-279. Beckett RP, Csintalan Z, Tuba Z. 2000. ABA treatment increases both the desiccation tolerance of photosynthesis, and non-photochemical quenching in the moss Atrichum undulatum. Plant Ecology 151: 65-71. Bray EA. 1997. Plant responses to water deficit. Trends in Plant Science 2: 48-54. Bukhor NG, Heber U, Wise C, Shuvalov VA. 2001a. Energy dissipation in photosynthesis: does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center? Planta 212: 749-758. Bukhov NG, Kopecky J, Pfiindel EE, Klughammer C, Heber U. 2001A. A few molecules of zeaxanthin per reaction center of photosystem II permit effective thermal dissipation of light energy in a poikilohydric moss. Planta 212: 739-748. Christianson ML. 2000. Control of morphogenesis in bryophytes. In: Shaw AJ, Goffinet B, eds. Bryophyte biology. Cambridge: Cambridge University Press, 199-224. Csintalan Z, Proctor MCF, Tuba Z. 1999. Chlorophyll fluorescence during drying and rehydration in the mosses Rhytidiadelphus loreus (Hedw.) Warnst., Anomodon viticulosus (Hedw.) Hook, and Tayl. and Grimmia pulvinata (Hedw.) Sm. Annals of Botany 84: 235-244. Deltoro V, Calatayud A, Gimeno C, Barreno E. 1998a. Water relations, chlorophyll fluorescence, and membrane permeability during desiccation in bryophytes from xeric, mesic, and hydric environments. Canadian Journal of Botany 76: 1923-1929. Deltoro V, Calatayud A, Gimeno C, Abadia A, Barreno E. 19986. Changes in chlorophyll a fluorescence, photosynthetic CO 2 assimilation and xanthophyll cycle interconversions during dehydration in desiccation-tolerant and intolerant liverworts. Planta 207: 224-228. Dietz S, Hartung W. 1998. Abscisic acid in lichens: variation, water relations and metabolism. New Phytologist 138: 99-106. Dietz S, Hartung W. 1999. The effect of abscisic acid on chlorophyll fluorescence in lichens under extreme water regimes. New Phytologist 143:495-501. Gaff DF. 1997. Mechanisms of desiccation tolerance in resurrection vascular plants. In: Basra AS, Basra RK, eds. Mechanisms of environmental stress resistance in plains. Netherlands: Harwood Academic Publishers, 43-58. Gilmore AM. 1997. Mechanistic aspects of xanthophyll cycle dependent photoprotection in higher plant chloroplasts and leaves. Physiologia Plantarum 99: 197-209. Halliwell B. 1987. Oxidative damage, lipid peroxidation and antioxidant protection in chloroplasts. Chemistry and Physics of Lipids 44: 327-340. Hartung VV, Schiller P, Diet/. KJ. 1998. Physiology of poikilohydric plants. Progress in Botany 59: 299-327. Heber U, Bilger \V, Bligny R, Lange OL. 2000. Phototolerance of lichens, mosses and higher plants in an alpine environment: analysis of photoreactions. Planta 211: 770-780. Hellwege EM, Dietz KJ, Hartung \V. 1996. Abscisic acid causes changes in gene expression involved in the induction of the landform of the liverwort Riccia ftuitans L. Planta 198: 423-432. Hellwege EM, Dietz KJ, Volk OH, Hartung W. 1994. Abscisic acid and the induction of desiccation tolerance in the extremely xerophilic liverwort Exormotheca holstii. Planta 194: 525-531. Hunt R, Parsons IT. 1974. A computer program for deriving growth functions in plant growth analysis. Journal of Applied Ecology 11: 297-307. Ingram J, Bartels D. 1996. The molecular basis of dehydration tolerance in plants. Annual Review of Plant Physiology and Plant Molecular Biology 47: 377-403. Ivanov AG, Krol M, Maxwell D, Huner NPA. 1995. Abscisic-acid induced protection against photoinhibition of PSII correlates with Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 constitutive tolerance is likely to be ancestral. We hypothesize that inducible tolerance mechanisms will be selected for in environments that are usually moist, and in which mosses only occasionally (and probably slowly) desiccate on a predictable seasonal basis. The latter corresponds to the habitat in which A. androgynum grows (Beckett and Hoddinott, 1997). We envisage that bryophytes with largely constitutive mechanisms will include species like Tortula that grow on exposed rocky surfaces. Although some induction of tolerance can apparently occur even in this genus (Schonbeck and Bewley, 1981a, b), rather than involving ABA, hardening seems to occur by the accumulation of 'recovery' mRNA transcripts into ribonuclear protein particles (Wood et al., 2000). It is worth noting that the overall desiccation tolerance of species with inducible tolerance mechanisms appears to be less than that of those with constitutive mechanisms. Comparing the tolerance to desiccation of photosynthesis in A. androgynum with that of other moss species (Proctor 2000«, b) suggests that it is rather sensitive. Further work is needed to assess the distribution of constitutive or inducible tolerance mechanisms in other bryophytes. ABA is clearly involved in many responses that harden the moss Atrichum to desiccation stress. Recently, Machuka et al. (1999) reported that ABA treatment induced the synthesis of at least eleven 'stress' genes in the moss Physcomitrella patens, although apparently these did not include genes involved with the xanthophyll cycle or with sugar metabolism. However, it should be noted that in higher plants (Bray, 1997) and bryophytes (Hellwege et al., 1994, 1996) ABA does not regulate all genes induced by drought or desiccation. In Atrichum, ABA pretreatment increases NPQ activity during the subsequent rehydration, and also slightly increases the concentrations of soluble sugars during desiccation. However, ABA does not affect starch metabolism or protect chlorophyll from breakdown during desiccation. Future progress in understanding the mechanism of desiccation tolerance in bryophytes will result from further studies of the gene products induced by ABA and other hardening treatments. In addition to the already investigated ABA- and desiccation-induced dehydrin-like proteins, the present study suggests that genes involved in the xanthophyll cycle are good targets for molecular investigation. 1099 1100 Mayaba et al.—Desiccation Tolerance of Photosynthesis in Atrichum androgynum and inhibitor experiments. Journal of Experimental Botany 51: 1695-1704. Santarius KA. 1994. Apoplastic water fractions and osmotic water potentials at full turgidity of some Bryidae. Planta 193: 32-37. Schonbeck MW, Bewley JD. 1981a. Responses of the moss Torlula ruralis to desiccation treatments. 1. Effects of minimum water contents and rates of dehydration and rehydration. Canadian Journal of Botany 59: 2689-2706. Schonbeck MW, Bewley JD. 1981A. Responses of the moss Tortula ruralis to desiccation treatments. I. Variations in desiccation tolerance. Canadian Journal of Botany 59: 2707—2712. Schreiber U, Bilger W. 1993. Progress in chlorophyll-fluorescence research: major developments during the past years in retrospect. Progress in Botany 54: 151-173. Schreiber U, Bilger W, Neubauer C. 1995. Chlorophyll fluorescence as a non-intrusive indicator for rapid assessment of in vivo photosynthesis. In: Schulze E-D, Caldwell MM, eds. Ecophysiology of photosynthesis. Berlin: Springer-Verlag, 49-70. Schwall G, Elster R, Ingram J, Bernacchia G, Bianchi G, Gallaher L, Salamini F, Bartels D. 1995. Carbohydrate metabolism in the desiccation tolerant plant Craterostigma plantagineum Hochst. In: Pontis H, Salerno G, Echeverria E, eds. Sucrose metabolism, biochemistry, physiology and molecular biology. Current topics in plant physiology, 14. Rockville: American Society of Plant Physiologists, 245-253. Scott P. 2000. Resurrection plants and the secrets of eternal leaf. Annals of Botany 85: 159-166. Seel WE, Hendry GAF, Lee JA. 1992. The combined effects of desiccation and irradiance on mosses from xeric and mesic habitats. Journal of Experimental Botany 43: 1023-1030. Smirnoff N. 1992. The carbohydrates of bryophytes in relation to desiccation tolerance. Journal of Bryology 17: 185-191. Smirnoff N. 1993. The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist 125: 27-58. Tuba Z, Csintalan Z, Proctor MCF. 1996. Photosynthetic responses of a moss, Tortula ruralis ssp. ruralis, and the lichens Cladonia convoluta and C. furcata to water deficit and short periods of desiccation: a baseline study at present-day CO 2 concentration. New Phytologist 133: 353-361. Werner O, Espin RMR, Bopp M, Atzorn R. 1991. Abscisic acid-induced drought tolerance in Funaria hygrometrica. Planta 186: 99-103. Wood AJ, Duff RJ, Oliver MJ. 2000. The translational apparatus of Tortula ruralis: polysomal retention of transcripts encoding the ribosomal proteins RPS14, RPS16 and RPS23 in desiccated and rehydrated gametophytes. Journal of Experimental Botany 51: 1655-1663. Zorn M, Pfeifhofer HW, Grill D, Kranner I. 2001. Responses of plastid pigments to desiccation and rehydration in the desert lichen Ramalina maciformis. Symbiosis 31: 201-211. Downloaded from http://aob.oxfordjournals.org/ by guest on October 5, 2015 enhanced activity of the xanthophyll cycle. FEBS Letters 371: 61-64. Kranner I, Lut/.oni F. 1999. Evolutionary consequences of transition to a lichen symbiotic state and physiological adaptation to oxidative damage associated with poikilohydry. In: Lerner HR, ed. Plant responses to environmental stresses. From phytohormones to genome reorganization. New York: Marcel Dekker, 591-628. Lichtenthaler HK. 1987. Chlorophylls and carotenoids, the pigments of the photosynthetic membranes. Methods in Enzymology 148: 350-382. McKersie BD, Lesham YY. 1994. Stress and stress coping in cultivated plants. Dordrecht: Kluwer. Machuka J, Bashiardes S, Ruben E, Spooner K, Cuming A, Knight C, Cove D. 1999. Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens. Plant and Cell Physiology 40: 378-387. Minibayeva FV, Beckett RP. 2001. High rates of extracellular superoxide production in bryophytes and lichens, and an oxidative burst in response to rehydration following desiccation. New Phytologist 152: 333-343. Niyogi KK. 2000. Safety valves for photosynthesis. Current Opinion in Plant Biology 3: 455-460. Niyogi KK, Grossman AR, Bjorkman O. 1998. Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conservation. The Plant Cell 10: 1121-1134. Oliver MJ. 1996. Desiccation tolerance in vegetative plant cells. Physiologia Plantarum 97: 779-787. Oliver MJ, Bewley JD. 1997. Desiccation-tolerance of plant tissues: a mechanistic overview. Horticultural Reviews 18: 171—213. Oliver MJ, Wood AJ. 1997. Desiccation tolerance in mosses. In: Koval T M , ed. Stress inducible processes in higher eukaryotic cells. New York: Plenum Press, 1-26. Oliver MJ, Tuba Z, Mishler BD. 2000. The origin of vegetative desiccation tolerance in land plants. Plant Ecology 151: 85-100. Oliver MJ, Wood AJ, O'Mahony P. 1997. How some plants recover from vegetative desiccation: a repair based strategy. Ada Physiologia Plantarum 19: 419-425. Oliver MJ, Wood AJ, O'Mahony P. 1998. To dryness and beyond': preparation for the dried state and rehydration in vegetative desiccation tolerant plants. Plant Growth Regulation 24: 193-201. Proctor MCF. 2000a. Physiological ecology. In: Shaw AJ, Goffinet B, eds. Bryophyte biology. Cambridge: Cambridge University Press, 225-247. Proctor MCF. 2000A. The bryophyte paradox: tolerance of desiccation, evasion of drought. Plant Ecology 151: 41-49. Proctor MCF, Smirnoff N. 2000. Rapid recovery of photosystems on rewetting desiccation-tolerant mosses: chlorophyll fluorescence