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Journal of Plant Physiology 162 (2005) 13311337

www.elsevier.de/jplph

Differential effects of abscisic acid on desiccation tolerance and carbohydrates in three species of liverworts
Valerie C. Pencea,, Susan S. Dunfordb, Steven Redellab
a

Center for Conservation and Research of Endangered Wildlife, Cincinnati Zoo & Botanical Garden, 3400 Vine Street, Cincinnati, OH 45220, USA b Department of Biological Sciences, University of Cincinnati, Cincinnati, OH 45221, USA Received 16 November 2004; accepted 10 February 2005

KEYWORDS ABA; Bryophyte; Desiccation; Marchantia polymorpha; Pallavicinia lyellii; Riccia uitans; Sugars

Summary
Tissues of three species of in vitro grown liverworts, Riccia uitans, Pallavicinia lyellii, and Marchantia polymorpha, were subjected to rapid drying with and without preculture for 1 week on medium containing 10 mM ABA. ABA preculture initiated total desiccation tolerance in R. uitans, whereas control tissues were killed after 30 min of drying. Survival was also improved in P. lyellii, whereas ABA did not affect survival of M. polymorpha after rapid drying. ABA treatment did, however, reduce the rate of water loss in M. polymorpha. Total soluble carbohydrates were increased in ABAtreated R. uitans and P. lyellii, but not in M. polymorpha, although there was no correlation between survival and changes in the percentage of these carbohydrates as reducing sugars. These differences in response to ABA and desiccation likely reect different adaptations of these three species to conditions in situ. & 2005 Elsevier GmbH. All rights reserved.

Introduction
Many plants, including byrophytes, possess morphological and physiological adaptations to extreme water decit. Among higher plants, desiccation tolerance is commonly found in seeds, spores and pollen, but it is less common among
Abbreviations: ABA, abscisic acid Corresponding author. Tel.: +1 513 569 8228; fax: +1 513 569 8213. E-mail address: valerie.pence@cincinnatizoo.org (V.C. Pence).

vegetative tissues. Many bryophytes, however, possess constitutive desiccation tolerance in their vegetative phase, while others can readily be induced to that state (Proctor and Pence, 2002). It is thought that a number of factors can contribute to desiccation tolerance in plants, including specic proteins, antioxidants, sugars and other compatible solutes (Buitink et al., 2002).

0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.02.002

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1332 The stress hormone, ABA appears to have a role in triggering factors involved in desiccation tolerance in a number of species, including bryophytes. ABA has been reported from Marchantia polymorpha (Li et al., 1994), as well as from 27 other bryophytes (Hartung et al., 1987), and the levels appeared to be correlated with natural desiccation tolerance. Slow drying of the protonemata of Funaria hygrometrica increased the endogenous levels of ABA (Werner et al., 1991), and desiccation tolerance in the liverwort Exomorthica holstii is correlated with increased levels of endogenous ABA and could be induced by the addition of exogenous ABA (Hellwege et al., 1994). While ABA appears to act as a trigger, an increase in certain sugars is one of the physiological changes that occurs in the tissues of several species as they acquire desiccation tolerance. Overall sugar concentrations have been shown to increase as desiccation tolerance increases in many plants (Amuti and Pollard, 1977; Leprince et al., 1990). In addition, the type of sugars also appears to be signicant, since a low ratio of monosaccharides to di- and polysaccharides has been found in several desiccation-tolerant species (Modi et al., 2000; Hoekstra and van Roekel, 1988; Koster and Leopold, 1988; Leprince et al., 1990). It has been hypothesized that non-reducing sugars, such as disaccharides and oligosaccharides, are the carbohydrates most directly involved in membrane stability and that high levels of reducing sugars are associated with a loss of desiccation tolerance. In the desiccation-tolerant moss Tortula ruraliformis, the concentration of reducing sugars decreased during desiccation (Smirnoff, 1992). The present studies were initiated to determine whether ABA can increase desiccation tolerance and change the carbohydrate composition in three species of liverworts: Riccia uitans, Pallavicinia lyellii, and M. polymorpha, all of which inhabit wet or moist areas. Total soluble carbohydrates and reducing sugars were measured both in ABA and non-ABA treated tissues. V.C. Pence et al. Shoot tips 1, 15, or 5 mm long were dissected from R. uitans, P. lyellii and M. polymorpha, respectively. In P. lyellii, however, two distinctive regenerative regions were used in survival experiments. Growth was recorded from both the shoot tips and the midrib, which included midrib initials, or ventral lateral primordia. Thus, the upper threequarters of the growing thallus, up to 1.5 cm, was used for harvesting P. lyellii.

Culture and media

Tissues were surface sterilized by 15 min of agitation in a 1:20 dilution of commercial bleach, containing Tween 20, one drop (approx. 0.05 mL)/ 50 mL of bleach solution, followed by two rinses in sterile puried water. Stock tissues were maintained on half-strength Murashige and Skoog (1962) basal salts with minimal organics (Linsmaier and Skoog, 1965), 1.5% sucrose, and 0.22% PhytagelTM. This medium was also used for recovery growth after desiccation. Where indicated, 10 mM ltersterilized 7ABA (Sigma Chemical Co.) was added to this medium after autoclaving. For pretreatment, tissues were transferred to ABA-containing medium for 7 days, as was done previously (Pence, 1998). Tissues were grown at 26 1C, under CoolWhite uorescent lights, 1030 mmoles m2 s1 PAR, 16 h light:8 h dark.

Desiccation experiments
Moisture loss and viability were measured for control and ABA-treated tissues of each species over a 2-h period of drying on sterile paper towels under the air ow of a laminar ow hood, with the pieces clearly separated from each other. Drying tissues were harvested at 15-min intervals and placed onto recovery medium or used for moisture determination, with 5, 58, and 410 pieces harvested at each time interval for M. polymorpha, P. lyellii, and R. uitans, respectively. Each piece of tissue was observed for survival at 7 and 14 days. Survival was expressed as the percentage of pieces with green, growing areas of tissue. Moisture content was determined on a wet weight basis for each treatment by weighing harvested tissues (wet weight) and then reweighing after 2430 h at 95 1C (dry weight). Four replicate experiments were done for each species for both moisture and survival.

Materials and methods

Plant material
Three liverwort species were used for experimentation. M. polymorpha L. was collected locally; P. lyellii (Hook.) Gray was collected in Red River Gorge in central Kentucky; and R. uitans L. was purchased from Carolina Biological Supply (Burlington, NC).

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Analysis of soluble sugars

Tissue was harvested with and without ABA pretreatment (0.11.0 g, depending on the species), and a portion of the sample was used for moisture determination to estimate the dry weight of the samples used for analysis. Tissues were extracted in 4 mL of boiling 80% ethanol. The extract was sonicated for 20 min, followed by centrifugation at 6000g for 10 min at 20 1C. The suprenatant was removed and the pellet resuspended in approximately 2 mL of boiling 80% ethanol and centrifuged as above. The supernatants were combined, and 20 mL of chloroform and 5 mL of 0.58% NaCl were added. The solution was mixed well and centrifuged at 3000g at 20 1C for 10 min. The ethanol was removed from the supernatant in vacuo at approximately 75 1C for approximately 15 min. The volume of the remaining aqueous extract was measured and brought up to 44.5 mL with water. Samples were stored at 20 1C until use. Total soluble carbohydrates were determined using the anthrone method (Yemm and Willis, 1954) and reducing sugars were determined using the Somogyi method (Somogyi, 1951), with glucose as a standard for both assays. Preliminary extractions from each species were made to determine the appropriate dilutions based on original fresh weights. Final sample extracts were thawed and diluted as indicated by these preliminary assays. Dilutions of extracts fell within the range of 0.0050.5 for the anthrone assay and 0.10.5 for the Somogyi assay, depending on the quantity of tissue used.

Statistical analysis
Data were analyzed for standard errors and ANOVA (TukeyKremer post-hoc test) using StatView 5.0.1.

Figure 1. Water loss during 2 h of drying from tissues of (A) R. uitans, (B) P. lyellii, and (C) M. polymorpha, with and without 1 week of preculture on 10 mM ABA. Asterisks indicate signicant differences between precultured and non-precultured tissues with the same drying time (Po0:01).

Results
The rate of water loss during drying was measured for each of the three species (Fig. 1). R. uitans rapidly lost water, reaching a moisture content of 11% after only 45 min of drying. Water loss from P. lyellii was not as rapid, and did not reach this level until after 120 min of drying. M. polymorpha, on the other hand, showed some conservation of moisture through the rst 15 min of drying, after which the rate of water loss increased, with moisture decreasing to 13% after

75 min. When tissues were treated with ABA, there was no change in the rate of water loss with R. uitans or P. lyellii, but water loss was signicantly slowed from ABA-treated tissues of M. polymorpha. Survival of untreated tissues of all three species decreased with drying (Fig. 2). R. uitans retained less than 20% viability after 15 min of drying which reduced moisture to 60% and showed no survival after 30 min of drying (Fig. 2), which corresponded to 25% moisture. Viability was lost more gradually with M. polymorpha and P. lyellii, which showed 70% and 90% survival at 60% moisture, respectively.

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1334 V.C. Pence et al. lateral buds of this species. There was no signicant effect of ABA on the survival of M. polymorpha after drying. Total soluble carbohydrates and reducing sugars signicantly increased in R. uitans and P. lyellii in response to treatment with ABA (Table 1), although no increases were observed in M. polymorpha. However, reducing sugars as a percent of the total soluble carbohydrates were not affected by ABA treatment in any of the species tested.

Discussion
Preculture on 10 mM ABA for 1 week improved survival after rapid drying in two of the three liverwort species tested. ABA-treated tissues of R. uitans showed 100% survival after rapid drying which proved lethal within 30 min without ABA preculture. This conrms previous observations that ABA pretreatment was necessary for the survival of R. uitans after the rapid desiccation used as a pretreatment for cryopreservation (Pence, 1998), and that ABA improved desiccation tolerance in submerged thalli of R. uitans (Hellwege et al., 1996). R. uitans is an amphibious member of a genus which is otherwise characterized by species adapted to soil habitats. Desiccation tolerance has been reported in R. macrocarpa, which was revived after 23 years of dry storage (Breuil-See, 1993). R. uitans, on the other hand, inhabits ponds as an aquatic organism, but can survive their periodic, gradual drying. It has been shown that endogenous ABA levels increase when R. uitans thalli are transferred from water to a moist, agar surface, a situation that also induced the landform morphology in this species and is similar to the slow drying encountered in nature (Hellwege et al., 1992). Our tissues also exhibited some landform morphological characteristics, including rhizoids and a compact habit. However, they were growing on PhytagelTM, rather than agar, and may not have been completely converted from the waterform to the landform physiology as evidenced by their sensitivity to desiccation. The drying rate in our experiments was also more rapid than that of Hellwege et al. (1996), and it may be that slower drying of our untreated tissues could result in improved survival. Nevertheless, ABA pretreatment induced 100% tolerance of rapid drying in R. uitans. In contrast, M. polymorpha and P. lyellii are matforming species adapted to growing on moist soils, and these lost water more slowly than R. uitans.

Figure 2. Survival following 2 h of drying under the laminar ow hood in tissues of (A) R. uitans, (B) P. lyellii midrib, (C) P. lyellii tips, and (D) M. polymorpha, with and without 1 week of preculture on 10 mM ABA. Asterisks indicate signicant differences between precultured and non-precultured tissues with the same drying time (Po0:01).

Most or all viability was lost after 90120 min of drying in these two species. In contrast, when tissues were precultured for 1 week on ABA, R. uitans showed 100% survival, even after 120 min of drying (Fig. 2). ABA also slowed the loss of viability in apices of P. lyellii, although it did not improve survival in the ventral

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Table 1. Total soluble carbohydrates and reducing sugars in three species of liverworts, with 1 week of preculture on medium with and without 10 mM ABA Species Total soluble carbohydrates (mg/g DW) Control R. uitans P. lyellii M. polymorpha 209734 201751 193753 ABA 697722 803740 13779 Reducing sugars (mg/g DW) Control 1473 1277 5279 ABA 7479 2971 5676 Reducing sugars (%) Control 6.771.1 6.371.2 29.174.7 ABA 10.671.3 3.670.2 40.773.4

Average of three determinations. Signicant differences (Po0:05) between control and ABA treatments within species.

These species are less likely to encounter predictable cycles of annual drying than R. uitans, and their growth habit provides a physical protection against rapid drying. In addition, the prominent midrib of P. lyellii also serves in water conduction and may provide an additional buffer against rapid water loss. ABA preculture improved desiccation tolerance in shoot tips of P. lyellii, with 75% viability retained after 120 min of drying to approximately 40% moisture, although ABA had no effect on lateral bud survival. M. polymorpha showed no increase in desiccation tolerance with ABA preculture, even though ABA is naturally found in this species (Li et al., 1994). Hellwege et al. (1994) have reported that the levels of endogenous ABA increase only slightly in desiccated M. polymorpha and not to the levels that correspond with the induction of desiccation tolerance in other species. There is some capacity for desiccation tolerance in M. polymorpha, however, since tissues have been shown to survive slow drying if they were encapsulated in alginate beads in addition to being precultured on ABA (Pence, 1998). When rapidly dried, the rate of water loss was slowed in ABA-treated tissues of M. polymorpha, a result similar to that observed with the fern, Polypodium virginianum (Reynolds and Bewley, 1993). This may represent an alternate strategy for survival, even though rapid drying may not be commonly encountered by M. polymorpha. Ventral scales may passively retard water loss, and ABA may have an effect on the air pores in this species. ABA induction of closure in stomata in sporophytes of hornworts and mosses has been reported (Hartung et al., 1994), and further studies on the effects of ABA on air pores in M. polymorpha could provide insights into the development of this process. Untreated liverworts in this study contained approximately 200 mg/g DW total soluble carbohydrates. This is slightly higher than measurements reported from several other bryophytes (Roser

et al., 1992; Smirnoff, 1992) and from leaves of desiccation-tolerant vascular plants (Ghasempour et al., 1998), although similar to that reported for Tortula ruralis (Bewley et al., 1978). However, ABA preculture increased total soluble carbohydrates in R. uitans and in P. lyellii, the two species that also showed an increase in desiccation tolerance with ABA preculture. In several systems, desiccation tolerance has also been correlated with a decrease in reducing sugars as a percent of the total soluble carbohydrates (Bertossi et al., 2001; Muller et al., 1997; Pukacka and Pukacki, 1997; Van Der Toorn and McKersie, 1995; Albini et al., 1994; Hoekstra et al., 1994; Koster and Leopold, 1988) although this is not always the case (Li and Sun, 1999). No signicant change in the percent of total soluble carbohydrates as reducing sugars was observed for any of the liverwort species examined in this study. The differences in response to desiccation and to ABA among these three liverworts likely reect their different adaptations and needs in situ. As with higher plants, ABA appears to be used as a trigger for inducing stress survival mechanisms, but these mechanisms vary with the different taxa. In the three liverworts examined here, responses to ABA ranged from total desiccation tolerance in R. uitans, to delayed damage in P. lyellii, to a slowing of the rate of drying in M. polymorpha. An increase in soluble carbohydrates was correlated with increased desiccation tolerance, although changes in the percent of carbohydrates as reducing sugars were not. It has been noted that bryophytes offer useful experimental models for the study of stress tolerance (Oliver et al., 2000b), in some cases showing constitutive desiccation tolerance not responsive to ABA, as in the case of the desert bryophyte T. ruralis, while in other cases exhibiting a non-constitutive response induced by ABA (Oliver et al., 2000a). The varied responses to ABA reported here for these liverworts from moist environments should provide insight into the variations in the mechanisms of desiccation tolerance and should also provide the basis for further

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1336 investigations into the adaptations of plants to the loss of water. V.C. Pence et al.
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Acknowledgement
The authors gratefully acknowledge the helpful comments of Dr. Jerry Snider in the course of this research.

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