122

After washing for 20 min in to ml of deionized distilled water, ex trace llular Cd2

.o- was displaced by shaking twice in 10 ml of20 mM NiCI2 for 30 min each. Disks were then washed for 30 min in deionized disti lied water, dried for 48 h al 80°C. weighed. and shaken for 1 h in 1M HNO~ to displace intrace llular Cd2+ and K+.

Cd1+ and K'" were determined by atomic absorption spectropho­tometry in an air/acety lene name.

Cd2+ uptake was measured at the start of the experiment (day 0), and half of the remaining disks (250) were siored at 100% rel­ative humidi ly (RH). The other 250 disks were placed at 53% RH [achieved using a saturated solution of Mg(NO,hl for one day. Ihen al 0% RH (achieved used si lica gel). Mler 1, 3, 5, 7 and 15 days, 50 of the disks siored at 0% RH were lransferred 10 53 % RH for I day and Ihen to 100% RH for I day. The rale of Cd" uptake was then measured in these disks and in the disks that had only been stored at 100% RH. Therefore. uptake was measured on days 0, 4, 6, 8, 10 and] 8 of Ihe experiment. Changes in the rdative wate r content (RWC) of disks treated in the same way as those desiccated for 15 days were measured in repl icate samples. RWC is here defined as:

Waler content R we = :;:-...:.c.:"",--,,-,==_ Turgid water C01!lent

Fresh weight - dry weight Turgid weight - dry weigh!

Turgid weight was determined by incubating lichen disks in deionized distilled water until no further increase in weight occurred (c. I h) .

The RWC of Ihe lichens rapidly dropped to c. 0.07 when they were transferred to an RH of 53%, and dropped 10 below 0.02 al an RH of 0% (Figure I A). Lichens recovered their previous RWCs after they were Iransferred from 0 10 53 to 100% RH, and then rehydrated in deionized distilled water. Desiccation stress did not damage cellular integrity. measured by the concentration of K-+ in the lichens, following Cd::!-+ uptake (Figure IB). Com­pared with other genera, Peltigera is a desiccation-sensitive

lichen. losing approximately 50% of its intracellular K + foll ow­ing sudden rewetling of dry plants (Buck & Brown 1979). In Ihe present experiment, following a more gradual rehydration , disks lost very lillIe K-+ into the treatment or displacement solutions (c.

I % of the tota l K-+ in all cases). Desiccation also had little effect on the rate of intracellular Cd" uplake (Figure I C). AI the end of the experimental period, desiccated di sks actually di splayed faster Cd2 ... uptake rates than material kept moist. Presumably, storing the lichens moist progressively damaged their metabo­lism, as Farrar (1976a) has already reported for Peltigera . Visual observations suggested that Peltigera growing at the collection site is periodically desiccated. Extracellular Cd2+ uptake was lower in desiccated marcrial , especially at the end of the experi­ment (Figure 1 D). In desiccated material, the dry weight of a rep­licate of len 6-mm disks decreased from 17 .5 ± 0.9 mg to 16.9 ± 1.5 mg, whereas in material kept moist. the weight increased to 19.9 ± 2.0 mg after 18 days. Allhough this increase was not sig­nificant . it does suggest that some growth look place during stor­age; possibly the new growth produced cell walls with a higher exchange capacity.

It is clearly important for lichens subjected to repeated wetting and drying cycles to have membrane-transporting systems that can recover and take up ions following desiccation. Results pre­sented here show that severe desiccation for periods of at least two weeks does not reduce the ability of even a desiccation­sensitive lichen to take up ions.

S. M r. J. Bot. 1996.62(2): 122- 125

Acknowledgements

The author gratefully acknowledges the FRD and the University of Natal Research Fund for fin ancial support.

References BECKETT. R.P. & BROWN, D.H. 1984 The con trol of cadmium uptake

in the lichen genus Pelligera. 1. expo BOl. 35: 107 1-1082. BLUM. O.B . 1974. Water relations. In : The Lichens , cds. V. Ahmadjian

& M.E. Hale. pp. 38 1-400. Academic Press. New York. BROWN. D.H. & BECKETr. R.P. 1985. Minerals and lichens: acqui si ­

tion, localisation and effects. In : Surface physio logy of lichens. cds. C. Vicente. D.H. Brown & M.E. Legaz, pp. 127-145. Univcrsidad Com­plutense Press, Madrid .

BUCK, G.W. & BROWN, D.H. 1979. The effect of desiccalion on cat­ion location in lichens. Ann. Bol. 44: 265- 277.

FARRAR, J.P. 1976a. Ecological physiology of the lichen HYf/ogYlTlnia phy.Hldes. I. Some effects of constant water saturation. New Phytol. 77: 93- 103.

FARRAR. J.P. 1976b. Ecologica l physiology of the lichcn HYJ10gYlTlnia phy.wdes. 11. Effec ts of we lt ing and drying cycles and the concept of 'physio logical buffering'. New Ph),lOl. 77: 105- 11 3.

HUNT, R. & PARSO NS LT. 1974. A computer program for deriving growth functions in plant growth analysis. J. appl. Ec:ol. II : 297-307.

KERSHAW. K.A. 1985. Physiological ecology of lichens. Cambridge UniverSity Press, Cambridge.

RUNDEL, P.W. 1988. Water relations. In: CRC handbook of lichenol­ogy. ed. M. Galun. Vo l. II. pp. 17-36. CRe Press , Boca Raton.

The water relations ofthe maritime lichen Roccella hypomecha (Ach.) Borg. studied using thermocouple psychrometry

R.P. Beckett NU Research Unit for Plant Growth and Development. Department of Botany, University of Natal Pietermaritzburg. Private Bag X01, Scottsville, 3209 Republic of Soulh Africa

ReL'eived I August 1995: revised 4 January /996

The water relations of the maritime lichen Rocce/la hypomecha were investigated using thermocouple psychrometry. Results showed that, while containing high concentrations of Na+, the osmotic potential and other aspects of the water relations of R. hypomecha resembled those of lichens growing in xeric non-saline habitats. The significance of these results for the water relations of maritime lichens is discussed.

Keywords: Desiccation, lichen. psychrometry, salinity stress, Ihermocouple.

The supralittoral zone of many rocky shores often possesses a distinct communily of maritime lichens (James el al. 1977). Little is known about Ihe physiology of these planls. Nash and Lange (1988) and Nash el al. (1990) showed that the sait sensi­tivi ty of a range of lichen species was proportional to thei r prox­imity to the sea. This suggests that the ability of maritime lichens to tolerate the large amounts of NaCI deposited by wave splash may be an important factor determining their distribution. NaCl will first , create osmotic effects, and second, effects due to the ions themselves. In response to the challenge of a salinity stress , many halophytic higher plants can display osmoregulation, i.e. can reduce their osmotic potential ('V1I) at full turgor (McKersie

S. Arr. 1. BOL. 1996. 62(2)

& Lcshem 1994). The advantages of this are first , that low I.Vlt

will reduce the rdative water content (RWC) at which turgor is lost. Second. low tVlI wi II reduce the water po tential of the plant at turgo r loss, thus creating a steeper gradient for the uptake of water from the soil. Preliminary observations of Feige (1972) and Hamada et al. (1994) showed that lichens can increase the concentrations of sugars or sugar alcoho ls in their tissues when exposed to solut ions of low lV. However, the \1111 of these lichens. and the values of 'tilt of maritime lichens collected from the field are unknown. Beckett (1 995) used the thermocouple psychromc. rer to show that lichens from exposed. dry habitats had lower osmotic potentials than plants from mesic sites, and as a result los t turgor at lower RWCs. However, no maritime lichens were induded in this survey. The objec ti ve of the present investigation was to examine the water relations of the maritime lichen Roc­celLa liypomec.:Ii{/. In particular, the aim was to test whether \Ill!

was significantly [ower than values obtained from other lichens.

Roccella hypomecha (Ach. ) Bory. was collected from a quartz­itic sandstone rocky shore in the supralittoral zone ncar Cape Town, South Africa. Plants were stored air dry for 4 days, then hydrated by first storing them at a relative humidity of 100% (in a desiccator over distiIIed water) at 20c e and a light intensity of 135 J1molcs m·2 s' \ for 2 days, then placing them in distiIIed water for 1 h. Lichens showed very little increase in weight after this time and were the refore assumed to be full y turgid .

Thallus wate r potential was determined using a Decagon SC-IOA thermocouple psychrometer linked to a Wescor RR-33T microvoltmeter. After equilibration for 4 h and measurement of c. 100 mg o f hydrated material, the plants were allowed to lose between 2 and 3 mg of water, and after 4 h, \V was again meas~

ured. This was repeated until 13 measurements had been made on each of five samples, and the plants had reached a RWC of c. 30% and a 'V of c. - 10 MPa. Standard solutions of known \V were always run with samples, and values of \if corrected to a temperature of 20°C.

Water in lichens can occur in the symplast. in the apoplast, i.c. the pores in the cell wall, and intercellularl y. i.e. between the cells. The symbols R" R(j' and Ri indicate the proportion of water in a fully hydrated thallus in these three fractions respectively. To estimate R" a pressure-volume (PV) curve, i. e. (- I /,~ ) as a func­Li on of (I-RWC), was drawn. The resulting curve was initially concave, but beyond the region where turgor is lost (i.e. where turgor no longer contributes to 4'). the curve became linear (Fig­ure IA). From the PV curve, turgor potential (\if p) was calculated as the ditference between the extrapolated linear portion of the curve and the actual curve. Nex t, \Vp was ploued as a function of RWC. Unlike higher plants. in Roccella hypomecha turgor did not start falling until the RWC fell to c. 0.67 (Figure IB). The water lost between 100% RWe and the RWC at which turgor started to fall was assumed to be intercellular, i.e. Ri• The RWCs for all the data were then recalculated to ex:clude intercellular water, i.e.

(Fresh WI-dry WI) R W C, = =--,-;--:---'7-'-='-'-'---='---~c;-;---,-~

(Tltrgid wI - dry wt - wt interce llular waler)

where RWC, is the relative water content corrected to exclude intercellular wate r. The PV curve was replotted (see Figure tA) and R" calculated from the X-axis intercept, then corrected back as a % of total thallus water. The osmotic potential at full turgor (\Vl!5) was calculated from the Y~ axis intercept of the linear por~ tion of the PV curve, i.e. the value of 4'11 at 100% RWC,_ (Tyree & Jarvis 1981). Turgor potentials were recalculated, and showed the expected decline with decreasing RWC (Figure IB). Tissue el asticity (Ev) was calculated from the relationship between

123

4

A

3 0---0 = before correction

____ = after correction

2

o 0.2 0.4 0.6 O.B 1.0

1 - RWC

2.0 B

0---0 = before correction 1.5 ________ = after correction

1.0

0.5

O+---~----.-~-'r----r----r----' 0.4 0.5 0.6 0.7

RWC

O.B 0.9 1.0

Figure 1 A. Pressure-volume curve of Roccella hypomecha before and after correction for intercellular water. B. Turgor potential as a function of RWC in R. tillctorum before and after correclion for apoplastic water. calculated as the difference between the ex.trapo~

lated linear portion of the lines in A and the fitted curve. Points rep­resent fitted values with 95% confidence limits, calculated using the spline program of Hunt and Parsons (1974).

turgor potential (ljIp) and RWC. (Stadel mann 1984).

The cellular location of cations and an index of desiccation tolerance was estimated by the method of Buck and Brown (1979). This involved keeping two sets of four replicales of 100 mg of lichen tissue at 0% and 100% relative humidity for 48 h. Sudden reweuing was simulated by shaking Lichen samples in 10 ml of distilled water for 0.5 h. They were then transferred to 10 ml of NiCI, for 0.5 h to displace cell wall-bound cations. Finally. they were shaken in 10 ml of I M RNO, for 1 h to

release intracellular ions. The concentrations of K +, Na+, Mg2+ and Cah in [he elutions were determined by atomic absorption spectrophotometry. For plants kept at 100% RH. this corre­sponded to the amounts of inter-, extra- and intracellular ions present in unstressed lichens. K+ leakage caused by desiccation was estimated as:

In.tracellular [K] lichens pretrea ted tOO %RH - 0 % RH itllraceliu/ar[K] lichens pretreated IOO%RH

[see Buck & Brown (1979) for discussion on the use of K"'"leak­age as an index of desiccation tolerance]. As a check on the esti­mate of W:u-derived from the PV curve, the \VX of five replicate samples was estimated as fo llows. Lichen tissue (100 mg) was rehydrated as described above and placed in the sample cups,

124

wrapped in at least three layers of Parafilm and immersed in liquid nitrogen for c. 5 min. The cups were allowed to warm to room temperature (c. 1 h). The Parafilm was then removed, and the sample cups rapidly transferred back to the thermocouple psychrometer. After an equilibration Lime of 1 h, \jJ was deter­mined. Assuming that freezing ruptures membranes and thus destroys turgor, 4' will equal \jJ1(. This method underestimates 'V1't

because intercellular and apoplastic water dilutes ions and mole­cules in the symplast. To correct for this, a modification of the equmion of Jones and Rawson (1979) was used:

W'D' = 1 R - R " ,

where \V1!k is the water potenrial of water-saturated killed lichens. The value of '+1 ,, < obtained in this way was in good agreement with that derived from the PV curve (Table I).

Tht! mean concentration of K+ (moles I-I ) in the cytoplasm was calculated as follows:

meu'1I IIJ/ra cellular rhallll.f [K] (mol N dry mass-I) x 1000

meal! thallus H2

0(J: K dry mas:, l) x (t _ R -R ,) " , The \ji lt of a soluti on of KCI of this concentration was determined

from rabies, and expressed as a percentage of \V:r t ' estimated as the mean of the values derived from the PV curve and the freez­ing methods.

Table 1 presents a summary of the results obtained in thi s

study. The thallus K+ concentration, the desiccation tolerance

measured as K" loss, and the characteristics of the water relations of R. hypomecha were similar to those of other lichens growing in dry microhabitats. In particular, they corresponded closely to values obtained from a population of R. montagne; growing 40 km inland. The main differences were that the maritime species had higher values of RIl and 6,. While this would nonnally mean

that turgor would be lost at higher RWCs (Radin 1983), the mar­itime species also had higher RI' As a result , plams lost turgor at similar RWCs to the non-maritime Roccella, and other xeric licht!ns. The low values of \111'(s' 6\, and RIl found in R. hypomecha enabled this lichen to maintain positive turgor down to a much lower RWC than values found in higher plants (Tyree & Jarvis 198 1). However, the lichen showed little evidence of osmoregu­lation in response to salinity; the value of \jI:rs (-2.4 MPa) was

S. Afc J. Bot. 1996.62(2)

Table 2 Cellular location of cations in un­stressed Rocce/la hypomecha determined using a modification of the method of Buck and Brown (1979)

K'

Na'

Intercellular ion Extracellular ion Intrace llu lar ion concentration

(J.lmoles g>l)

20± I

8±1

42 ± 8

611 ± 76

concentration

(J.lmoles g- l )

I±O

M± 11

14± I

38 ± 5

concentration

(l1moles g-l)

20 ± I

869 ± 75

9±1

15 ± 5

very similar to that of the inland species, and much higher than values recorded in some halophytic higher plants (McKersie & Leshem 1994).

The cellular location of cations in unstressed lichens is pre­sented in Table 2. R. hypomecha had high intracellular concentra­tions of Ca1+. Although it is tempting to speculate that these derive from the trapping of Ca2+-rich particles in the thallus, the lichens were collected from quartzitic sandstone. Possibly the high values represent deposits of Ca1+ retained in the thallus as Ca2 .. oxalate by a process of secondary deposition (see Brown

1987 for review). K' only contributed about 7% to 1jI., (Table I) ,

suggesting that organic molecules were responsible for most of the'l'lu- No attempt was made to identify these molecules in this study, but recent surveys of lichens suggest that they contain high concentrations of sugars and polyols (Roser et af. 1992). These sugars may protect membranes from the high concentrations of ions that occur in desiccated ti ssues (Bewley & Krochko 1981; GalT 1989).

The lichens contained Na+ in both inter· and ex tracellular loca­tions, presumably as a result of continual splashing with sea­water and deposition of aerosols (Table 2). However, the plants

contained little cytoplasmic Na+. suggesting that the lichens pos­sessed a Na+ exclusion mechanism . Calculations revealed that \V:r at turgor loss was -S.4 ± 1.9 Mpa. Therefore, theoretically, these

plants could take up water from sea-water (1jI ~ -2.4 MPa). How­

ever, based on the intercellular concentration of Na+ (Table 2)

Table 1 Thallus K contents, K lost on rewetting and characteristics of the water relations of Roccella hypomecha. Data for terrestrial lichens from xeric sites and R. montagneitaken from Beckett (1995). Figures are given ± one standard deviation, n ~ 5; • excludes intercellular K

K+ can· Thallus Thallus K" 1jI, (MPa) 1jI,(MPa) centralian H,o rntercel- Apoplas-

content K"lost on {estimated (estimated Mean of eyto- content lular thai- tie thai· E,. at \VI' RWC {Jlmoles g rewelting from PV from 1jI. plasm \jilt due to (g g dry Ius H2O Ius H2O = I MPa at turgor

dry mass-I )* (% total) curve) freezing) (MP.) (mM) K' (%) mass· l ) (% total) (% 10.,1) (MP.) loss

ROI..:celfa 20 ± I 11.5 ± 5.4 -2.19 ± 0.59 - 2.62 ± O.IS -2.40 56 ± 4 7.t ± 0.5 0.79 ± 0.02 33 ± 5 23 ± 5 5.3 ± 5.4 42±6 hypomec:ha

Mean of four 73 20 -2.36 - 2.36 -2.36 89 17 1.1 7 19 13 1.8 45 xeric species

Rocc:ella 29 7 - 2.79 -2.88 -2.84 50 8 0.88 14 14 1.8 43 montagnei

(xeric non-maritime species)

S. Afr. J . Bot.. 1996,62(2)

and the t!stimate of the amount of intercellular water in a turgid lichen (R" Table 1), and assuming that the Na+ was present as

Nnel , the osmotic potent ia l of the intercellu lar wate r at 20°C would be - 11.4 ± 1.3 MPa. Wetting the lichen with waler may dissolve extrace llular Nael, thus creating a bathing solution with a very low \!/II~' capable of plasmolysing and damaging the cell s.

It seems likely therefore that R. hypomecha usually becomes hydrated by atmospheric moisture, which would be abundant ncar th !.! sea. This is consistent with the observation of Lange et al. (1989) that lichens like Roccel/a with chlorophycean photo­bionts can achieve net photosynthesis from atmospheric moisture alone.

Acknowledgements

I gratefull y acknowledge the Foundation for Research Develop­ment and the University of Natal Research Fund for financial support and Pra rT.H. Nash III for passing valuable comments on the manuscript.

References BECKETI'. R.P. 1995. Some aspects of the water relations of lichens

from contrasting habitats stud ied usi ng thermocouple psychrometry.

Ann. BOT. 76. 2 11-2 17. BEWLEY, J.D . & KROCHKO, J.E. 1981 . Desiccation tolerance. In:

Em:ydopaedi<J. of plant physiology. New series . Volume 128. Phys io­logica l plant ecology II. Water relations and carbon assimilation. cds. C.L. Lange. P.S. Nobel. e.B Osmond & H. Ziegler. pp. 325-378. Springer-Verlag. Berlin.

BROWN, D.H. 1987. The location of mineral elements in lichens: impli ­

cations for metabolism. BilJlphy Lic:helloi. 25: 361 - 375. BUCK. G.W. & BROWN, D.H. 1979. The effect o f desiccation on cal­

ion location in lichens. Anfl. Bot. 44: 265-277. FEIGE, G.B. 1972. Ecophysiological aspects of carbohydrate metabo·

lism in the marine blue green algae liche n LiI.:J/ina {Jygmaell AG. Z PflPhy.<;ioi.68: 121- 126.

GAFF, D.E 1989. Responses of desiccation tolerant 'resurrection' plants to water stress. In : S tructural and functional responses to environmen­tal stress: water short age. cds . K. H. Kreeb. H. Ri chter & T.M. Hinck­ley. pp. 255- 268. SPB Academic, The Hague.

125

HAMADA. N .. OK AZAKl. K. & SHINOZAKI. M. 1994. Accu mula­tion of monosaccharides in lichen mycob io nts c ultured under osmot ic conditions. Byn l/ogisI 97: 176-179.

HUNT, R. & PARSONS, I.T. 1974. A computer program fo r deriving growth functions in plant growth analys is. J. app. Ew/. 11: 297- 307.

JAMES. P.W. HAWKSWORTH. D.L. & ROSE. F. 1977. Lichen com­munities in the British Isles: A preliminary conspectus . In: Lichen eco logy, ed, M.R.D. Seaward , pp. 295-4 13. Academic Press, London.

JONES, M.M. & RAWSON, H.M. 1979. Innuence of rate of develop­ment of leaf waler deficits upon photosynthesis, leaf conductance. water use efficiency, and osmotic potenti al in sorghum. Physi%gia PI. 45: 103- 111.

LANGE. O.L.. BILGER. W .. RIMKE. S. & SCHR EIBER. U. 1989. Chlorophyll fluorescence of lichens containing green and hlue-green algae during hydration by water vapor uptake and by addition o f liquid water. Bor. Acta 102: 303-313.

McKERSIE, B.D. & LESHEM. Y.Y. 1994. S tress and stress coping in c ultivated plants. Kl uwer Acade mic Press, Dordrecht.

NASH, T.H. & LANGE. O .L. 1988. Responses o f liche ns (0 sa linity: concentration and time-course re lationsh ips and variability among Cali forn ian species. New Phytol. 109: 36 1-367.

NASH . T.H .. REINER . A .. OEMMIG -A DAMS. B .. KILIAN. E .. KAI­SER, W.M. & LANGE. O.L. 1990. The effect of atmospheric desicca­tion a.nd osmotic water stress on photosynthesis and dark respiration of lichens. New Phytol. 116: 269-276.

RAD IN. J.W. 1983. PhYSiological consequences of ce llular water defi­cits: osmolic adjustment. (n: Limitations to effic ient water use in crop production, eds. H.M. Taylor. W.R. Jordan & T. R. Sinclair, pr. 267-276. American Society of Agronomy. Wiscons in.

ROSER. OJ .. MELICK. O.R .. LING, H.U. & SEPPELT R.D. 1992. Polyol and sugar content of terrestrial plants from continental Antarc­lica . AlltarC:lic: Sci. 4: 413-420.

STAOELMANN, E.J. 1984 The derivation of the ce ll wall elasticity func tion from the cell turgor potential. J. expo Bot. 35: 859-868.

TYREE, M.T. & JARV IS, P.G. 1981. Water in t issue and cells. In: Ency­

clopaedia of plant physiology. New series. Vo lume 12B. Physiolog ical plant eco logy II. Water relations and carbon ass imilation. cds. O.L. Lange, P. S. Nobel. e.B Osmond & H. Z ieg ler, pp. :'5- 77. Springer-Verlag. Berlin.