Planta (1992)187:282 294
P l a n t a (~) Springer-Verlag 1992
Carbon-dioxide exchange in lichens: determination of transport and carboxylation characteristics I.R. Cowan 1, O.L. Lange*, and T.G.A. Green2 Lehrstuhl ffir Botanik II der Universitfit W/irzburg, Mittlerer Dallenbergweg 64, W-8700 Wiirzburg, Federal Republic of Germany Received 8 October 1991; accepted 11 January 1992
Abstract. Measurements were made o f net rates of CO2 assimilation in lichens at various ambient concentrations o f COs in air and in helox (79% He, 21% 02). Because o f the faster rate o f CO2 diffusion in the pores of lichen thalli when filled with helox than when filled with air, a given net rate of assimilation was achieved at a lower ambient concentration o f CO2 in helox. The differences were used to estimate resistances to diffusion through the gas-filled pore systems in lichens. The technique was first * To whom correspondence should be addressed 1 Permanent address: Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia 2 Permanent address: Department of Biological Sciences, University of Waikato, Hamilton, New Zealand Symbols and units: Rates of assimilation, carboxylation, and respiration are expressed as molar flux of CO2 per mole of chlorophyll (s-1); concentrations as volume COs per unit volume carrier gas (dimensionless); and resistances as concentration differenceper unit rate of assimilation or carboxylation (s).
a = rate of CO2 diffusion in air relative to that in carrier gas (unity if the carrier gas is air and 0.43 if is helox); Al=net rate of COz uptake by the lichen; Ap=gross rate of carboxylation minus photorespiratory decarboxylation in the phycobiont, i.e. net rate of light-activated CO2 exchange; A, = maximum, CO2-saturated magnitude of Ap; c=concentration of CO2; c,=ambient concentration of CO2; ci=c~ minus difference in CO2 concentration across air-filled pore space in the thallus; c~=COz concentration equivalent to partial pressure of CO2 at the surface of the phycobiont; F1 = magnitude of ca at which A1 = 0; F. = magnitude of c. at which Ap = 0; R = rate of "dark respiration" in the lichen (mycobiont and phycobiont); R'= rate of "dark respiration" in region between the surface of the lichen and an arbitrary distance from the surface within the thallus; r = resistance to CO2 transfer from lichen surface to the surface of the phycobiont; rt = resistance to CO2 transfer between effective source of "dark respiration" in the lichen and the surface of the phycobiont; r,, r~=components of r and r*, respectively, attributable to transfer in air-phase; r,~., r~,=comportents of r and r*, respectively, attributable to transfer in waterphase; if=component of r between surface of lichen and an arbitrary distance from the surface within the thallus; r, = resistance to CO 2 transfer and carboxylation in the phycobiont; RH=relative humidity
tested with five lichen species, and then applied in a detailed study with Ramalina maciformis, in which gasphase resistances were determined in samples at four different states of hydration and with two irradiances. By assuming, on the basis o f previous evidence, that the phycobiont in R. maciformis is fully turgid and photosynthetically competent at the smallest hydration imposed (equilibration with vapour at 97 % relative humidity), and that, with this state of hydration, diffusion o f CO2 to the phycobiont takes place through continuously gas-filled pores, it was possible also to determine both the dependence of net rate o f assimilation in the phycobiont on local concentration o f CO2 in the algal layer, and, with the wetter samples, the extents to which diffusion o f CO2 to the phycobiont was impeded by water films. In equilibrium with air of 97 % relative humidity, the thallus water content being 0.5 g per g dry weight, the resistance to CO2 diffusion through the thallus was about twice as large as the resistance to CO2 uptake within the phycobiont. Total resistance to diffusion increased rapidly with increase in hydration. At a water content o f 2 g per g it was about 50 times as great as the resistance to uptake within the phycobiont and more than two-thirds o f it was attributable to impedance of transfer by water. The influences of water content on rate of assimilation at various irradiances are discussed. The analysis shows that the local CO 2 compensation concentration o f the phycobiont in R. maciformis is close to zero, indicating that photorespiratory release o f CO 2 does not take place in the alga, Trebouxia sp., under the conditions of these experiments.
Key words: Carbon dioxide (diffusion and carboxylation resistance) --Helox - Lichen - Photosynthesis (lichen) Water content (lichen)
Introduction Symbiosis in a lichen seems to be an awkward partnership. The initiation and maintenance of metabolic activ-
I.R. Cowan et al. : Carbon-dioxide exchange in lichens ity in phycobiont and mycobiont requires that water be taken up and stored in the lichen thallus, yet the swelling of the fungal hyphae, and the blocking of pores within the matrix of hyphae by extracellular water, consequent on hydration, impedes the diffusion o f COz to the phycobiont (Lange and Tenhunen 1981; Snelgar et al. 1981a; see Kershaw 1985). Our understanding of the ecophysiology of lichens would be m u c h improved if the effects of hydration on metabolism and physical resistance to CO2 diffusion could be analysed individually, but the effects have proven difficult to separate. As water is freely evaporated f r o m the entire surface o f the thallus exposed to the atmosphere, measurement o f v a p o u r loss cannot be used, as it can with leaves o f higher plants, to provide information relevant to the passage of CO2 into the plant tissue. Green and Snelgar (1981) attempted to determine resistances to COz transfer in Stieta lat~'ons and Pseudocyphellaria amphisticta (= P. tividofusca) by analysing the dependence of net rate of assimilation of COz, A1 in each lichen on ambient CO2 concentration ca, having assumed a particular f o r m for the dependence o f net rate o f assimilation in the phycobiont, Ap on the local concentration experienced by the phycobiont % There is some circularity in the logic of this approach, as the f o r m of the function Ap (cs) is a property one wishes to determine without presumption. Later, Snelgar et al. (1981 b) estimated resistances to CO: transfer in the same species using measurements of the a n a t o m y of the cyphellae and pseudocyphellae, and obtained rough agreement with the previous estimates with S. latifrons, but p o o r agreement with P. amphisticta. In this article we describe a new approach to the problem: the use o f a technique previously exploited to study gaseous diffusion processes in animals, and in higher plants (Egorov and K a r p u s h k i n 1988; Parkhurst and M o t t 1990). The relationships A1 (ca) were determined in both air and in helox (79% He, 21% Oz). As CO2 diffuses 2.3 times faster in helox than in air, any given rate of assimilation is achieved with a lower concentration of CO2 in helox than in air. The differences in concentration were used to determine the resistances to gas diffusion in the lichen thalli. We describe b o t h exploratory measurements with five lichen species, and a more comprehensive investigation with Ramalina maciformis at four different water contents and two light intensities. Ramalina maciformis is a particularly appropriate species to use, not only because it has been the subject of m a n y ecophysiological studies (e.g. Lange 1969; Lange et al. 1970; K a p p e n et al. 1979). The green algal phycobiont becomes fully turgid (Btidel and Lange 1991) and appears to be fully competent photosynthetically (Lange et al. 1989) when the lichen is moistened by equilibration with v a p o u r at a relative humidity ( R H ) o f only 97%, equivalent to - 4 0 bar water potential. A t this pressure, only pores having radii less than a b o u t 4 gm are filled with liquid water, and it is reasonable to assume that the thallus contains a continuously gas-filled pore system extending f r o m the external surface to the algal layer. Therefore, the entire resistance to CO2 diffusion (except that in the phycobiont) m a y be estimated by comparison of assimilation characteristics in air and
283 helox, and used to determine how the carboxytation rate in the phycobiont responds to variation in local C O / concentration.
Materials and methods
Material. Preliminary measurements were made with one liverwort and five species of lichens, followed by a more comprehensive investigation with the lichen Ramalina maciformis. The Marchantiales liverwort, Monoclea forsteri Hook., was collected from a native evergreen forest in the Hakarimata Ranges near Ngaruawahia, New Zealand, and kept wet until, and during, the course of the experiments. The foliose lichen species Pseudo~vpheltariafaveolata (Delise) Malme (green phycobiont), P. cotensoi (Church. Bab.) Vainio (green phycobiont), Sticta fuliginosa (Dickson) Ach, (bluegreen phycobiont), S. latifrons A. Rich. (green phycobiont) were collected at Mt. Tarawera near Rotorua (New Zealand), from regenerating forest on the rift plateau. The bushy-fruticose R. maciformis (Del.) Bory (green phycobiont), which is typical of the Negev Desert, was collected near Shivta (Israel). The lichen thalli were transported in air-dry conditions and subsequently stored at low temperatures. Some characteristics of the samples used are given in Table I. Chlorophyll contents were determined according to Ronen and Galun (1984). Apparatus, The equipment, which measures CO2 exchange by samples in each of four cuvettes with controlled COz concentration, temperature and water-vapour pressure, is fully described in Lange and Redon (1983) and Lange et al. (1986). Helox was provided in tanks, the oxygen content of each mixture having been verified with an oxygen electrode. The infra-red absolute and differential COz analysers (Binos, Leybold, Hanau, FRG) were calibrated, with air and helox as the carrier gases, using mixing pumps (Digamix, W6sthoff, Bochum, FRG; see Beyschlag et al. 1986). The sensitivity of the instruments was 5-10% less with helox than with air in both absolute and differential modes of measurement, the difference in the absolute mode increasing with CO2 concentration. During both calibration and measurement, all gas streams entering the IRGA were first brought to uniform concentration of water vapour by humidification at room temperature followed by dehumidification at 2~C in a Peltier-operated vapour trap. In this way errors due to any sensitivity of the analyser to water vapour were avoided. All mass-flow meters and mass-flow controllers (Tylan, Carson, Calif., USA) were also calibrated for air and for helox. Their sensitivity was about 30% greater with helox than with air, corresponding to the difference in the heat capacities per unit volume at constant pressure of the two mixtures. Finally, the accuracy of the complete operational gas-exchange measurement system was tested by use of a small mass-flow controller (Tylan) to inject air or helox into one of the measuring cuvettes to simulate the dilution of CO2 in the Table 1. Species and characteristics of samples
Species
Mass/unit area (g' m -2)
Chlorophyll/ unit area (mmol. m -z)
Monoclea forsteri Pseudocyphellariafaveolata Pseudocyphellaria colensoi Sticta fuliginosa Sticta latifrons Ramalina maciformis Ramalina rnaciformis (O) Ramalina maeiformis (A)
44 75 14I 103 114 264 214 223
0.38 0.28 0.27 (0.31)~ 0.34 0.55 0.28 0.35
For symbols see Fig. 5 " Not known: chlorophyll per unit mass was taken as that in S. tatifrons
284 main gas stream brought about by assimilation in a lichen sample. The system estimated apparent rate of assimilation accurately when CO2-free air was added to air streams having various CO2 contents, but underestimated it by 3 % when pure helox was added to streams of helox having various CO2 contents. Allowance was made in all experiments for this discrepancy, the cause of which was not identified. In order to estimate the resistance to CO2 transfer of the boundary layer between the ambient gas and the lichen surfaces, the rate of evaporation from a wet filter paper having approximately the area of the samples to be studied (30 cm2), placed in one of the cuvettes, was measured. The boundary-layer resistance to vapour transfer per unit area was found by applying energy-balance theory to calculate the temperature of the paper (Cowan and Farquhar 1977), and was less than 2 m 2 9s 9mol-x, equivalent to a resistance to CO2 transfer of 3 m 2 9 s 9m o l - t. The smallest resistances to CO2 transfer per unit area within and from lichens during photosynthesis were estimated to be 30 m z 9s 9m o l - ~ and therefore the characteristics of air flow in the cuvettes can have had little effect on CO2 assimilation rates and CO2 concentrations in the lichens. Figure 2 shows measurements of assimilation rates in a sample of the liverwort, M. forsteri, having about the same capacity for carbon fixation as the lichen samples but, unlike lichens, containing no internal airspace. That the assimilation characteristics in air and in helox were identical indicates that the resistance of the boundary layer was indeed negligible.
Experimental procedure. The lichens used in the first experiment were activated by wetting the thaUi and keeping them at 10~ C with a 12:12 h light:dark cycle for 4 d before commencing measurements. They were then rewetted by spraying with liquid water, the excess water being removed by blotting the undersides of the thalli with tissue paper (Snelgar 1981), and placed in the cuvettes. The two samples of R. maeiformis used in the more detailed experiment were initially activated by humidification at 97 % RH in the experimental cuvettes for 56 h. Following measurements of CO2 exchange at this state of hydration, further measurements were made with one of the samples having been wetted by immersion and the other having been infiltrated with water under vacuum (water-jet pump). A third set of measurements was made with the water content of the first sample reduced by blotting and then increased by light spraying, and that of the second sample maintained by re-infiltration. The water contents corresponding to the four conditions of hydration, vapour-equilibrated, lightly wetted, copiously wetted and infiltrated, were about 0.5, 1.2, 2 and 6 g per g dry weight, respectively. Water loss from the illuminated lichen samples during the experiments was unavoidable, but was minimized by supplying air of 97 % RH to the cuvettes. Rate of assimilation was especially sensitive to changes in water content of the thalli when in quasi-equilibrium at this humidity, and therefore each measurement in the light (which took 20 min) was followed by a period of 3 to 4 h darkness in which loss of water was made good by vapour re-equilibration. Moistening of the lightly wetted sample was repeated in the course of measurements and the small changes in water content brought about may have been responsible for some scatter in the data obtained. Measurements of net rate of assimilation at various ambient concentrations of CO2 in air and in helox were made with each lichen (Fig. 2), and, with R. maeiformis, in each state of hydration (Fig. 3). Photosynthetically active irradiance was 120 lamol- m - 2 - s - t in the preliminary experiments, except with R. maeiformis which received 300 ~tmol 9m - 2. s- 1. These irradiances were sufficient to induce maximum rates of photosynthesis with ambient concentrations of CO2 in the range 0 to about 300 lal 91-1. The more detailed measurements with R. maciformis were carried out with two irradiances: 105 and 300 lamol' m -2" s -x. Cuvette temperature was 15.0 4- 0.1 o C in all experiments. Concentrations of CO2 were step-changed in the range 0 to about 350 ~tl - 1-t with some measurements (data not shown) being made at concentrations up to 2,500 ~tl 91-1. During the course of each set of measurements, frequent switches were made between air
I.R. Cowan et al.: Carbon-dioxide exchange in lichens and helox as the carrier gas. Following each change of C O 2 c o n and-or carrier gas, an interval of at least 15 rain was allowed to elapse before the subsequent measurement was made to ensure that a steady rate of assimilation had been achieved. The completion of a set of measurements was most protracted (72 h) with R. maciformis in the vapour-equilibrated state. Measurements of rate of respiration in the dark were made for each lichen sample and each state of hydration. Rates in air and in helox were identical, as were the net rates of assimilation at a given irradiance with a saturating concentration of CO2. Therefore we concluded that the substitution of helox for air did not directly affect either respiratory or photosynthetic metabolism of the lichens and that all effects caused by change of carrier gas could be attributed to differences in the speeds of CO2 diffusion in the lichen thalli. centration
Theory Our experiments had three interrelated objectives: to d e t e r m i n e r e s i s t a n c e s to C O 2 t r a n s f e r in l i c h e n thalli, e s p e c i a l l y as a f f e c t e d b y w a t e r c o n t e n t ; to e s t i m a t e l o c a l c o n c e n t r a t i o n s o f C O 2 in t h e v i c i n i t y o f t h e a l g a l p h y c o b i o n t s ; a n d to e x a m i n e t h e d e p e n d e n c e o f C O 2 f i x a t i o n r a t e s in t h e p h y c o b i o n t s o n l o c a l c o n c e n t r a t i o n o f C O 2 . I n Fig. 1a t h e f l o w p a t h o f C O 2 f r o m a m b i e n t a t m o s p h e r e , in w h i c h t h e c o n c e n t r a t i o n is ca, to t h e s u r f a c e o f t h e p h y c o b i o n t in t h e a l g a l layer, w h e r e t h e c o n c e n t r a t i o n is % is r e p r e s e n t e d b y a single resistor. I f t h e n e t r a t e o f C O 2 u p t a k e b y t h e l i c h e n is A1 a n d t h e n e t r a t e o f d a r k r e s p i r a t i o n in b o t h m y c o b i o n t a n d p h y c o b i o n t is R , t h e n A t + R = Ap, say, is t h e g r o s s r a t e o f c a r b o x y l a t i o n m i n u s p h o t o r e s p i r a t i o n in t h e p h y c o b i o n t . I n g e n e r a l , d a r k r e s p i r a t i o n is r e l e a s e d at v a r y i n g r a t e s a l o n g t h e e n t i r e f l o w p a t h , b u t it is c o n v e n i e n t to d e f i n e a single p o i n t at
a A~
AI*_~R'
Ap
s-O I
r'
r - r'
i t I I
t I t
R'
AIj,.
i
R-R'
I
A I+._.R
._~,..: AI +R
/
rg - rg*
rw - rw* I
rg~
rw'
/R Fig. la, b Scheme of fluxes and resistances used to describe diffusion of CO2 in a lichen from external atmosphere (left) to phycobiont (right). In a, the catena of resistances is arbitrarily divided in two, the resistance components being r' and r-r'. The flux at the dividing point in the catena &resistances is intermediate between the net rate of assimilation of the lichen (At) and that of the phycobiont tAp) being the sum of At and the rate of respiration (R') in the portion of thallus external to that point. In b, the catena is split in two at the effective centre of respiration rate (R) in the thallus and further subdivided into gas-phase and water-phase components, distinguished by subscripts g and w
I.R. Cowan et al.: Carbon-dioxideexchangein lichens
285
which it is effectively all evolved. Let the rate of respiration released into a segment of the flow path, resistance r', extending from the surface to any arbitrary distance in the thallus be R'. Then A~ + R' = - d c / d r ' , and by integration over the whole path, resistance r, from surface of the lichen to the surface of the phycobiont it follows that ca = A l r + R r t + c ~
(Eq. 1)
Ap= AI+R-
where 1 /'r r* = ~ [ R ' d r ' J 0
The resistance rt has a maximum of r, which would obtain if all of the respiration took place at the surface of the thallus, and a minimum of zero, which would obtain if all of the respiration took place within, or at the surface of the phycobiont. If respiratory COz were evolved uniformly along the resistance path, for example, then R ' = r'R/r, leading to r* = r/2. In general, r* defines the position of the "centre of gravity", as it were, of dark respiration. Therefore the system can now be represented by Fig. lb, with the resistances in the gas-phase and water-phase components of the flow path being distinguished by subscripts g and w respectively. The gas-phase in the lichen normally consists of air, of course. If the lichen is placed in helox, account must be taken of the change in diffusion rate of CO2. In general, the relationship between CO2 transport and concentration difference is described by the equation ca = A1 (err,+ rw) + R (ctrtg+r~) + cs
(gq. 2)
in which a is unity with air and 0.43 with helox. As the carbon metabolisms of mycobiont and phycobiont are not affected by the substitution of helium for nitrogen, the concentration c, corresponding to any particular net rate of assimilation, A1, attained with a particular set of environmental conditions will be independent of the carrier gas used. However, the ambient concentration of COz required to achieve that rate of assimilation will be greater with air than with helox because of the smaller diffusivity of CO2 in air. From Eq. 2, the difference is
kca = 0.57" (A~rg+ Rrgt)
helox to give exactly the same net rates of assimiliation. Interpolation is required. It is desirable that the mathematical function used be consistent, not only with the model in Fig. lb, but also with a plausible description of the assimilation characteristics of the phycobiont. We express the latter in the form of a Michaelis-Menten response to CO2 concentration with allowance made for photorespiration, i.e.
(Eq. 3),
the numerial factor being the difference between a for air and for helox. Thus Aca is a linear function of A1 for constant conditions of thallus water content, temperature, and irradiance. The resistance rg is defined by the slope, and, provided R may be equated with rate of respiration measured in the dark t, the resistance rg* is defined by the intercept at A1 = 0. In practice, however, Ac, is not found directly, as it is inconvenient to adjust concentrations of CO2 in air and It seemsunlikelythat respiration in the mycobiontis substantially suppressed in the light(see Kappenand Lange 1972). Recentstudies with a higher plant (McCashin et al. 1988) and with algae (Weger and Turpin 1989; Turpin 1991)indicate that dark respiration in the phycobiont may also be expected to continue essentiallyundiminished in the light, at least over the range of illuminationused in the present experiments.
cs- F , r, + c J A ,
(Eq. 4)
where F , is the magnitude of c~ at which Ap = 0 (i.e. at which rate of gross carboxylation exactly balances rate ofphotorespiration), r, is the resistance to C02 transport and carboxylation in the phycobiont, and A , is the maximum, C02-saturated magnitude of Ap. Using this equation to eliminate cs in Eq. 2, it follows that ca = Al(~rg+rw)+R (arg+ * rw+ * F./R) +
(A1 + R) (r, § F , / A , )
1 -- (At + R ) / A ,
(Eq. 5)
This is a quadratic equation in At and is readily inverted to express A1 as a function of the two quantities, Ca and a, that are varied in each experiment - the water content, temperature, and irradiance of the lichens being constant. It may be fitted to the data, using an algorithm minimising the mean square deviation in A1. If R is known the process yields estimates of six parameters: rg, r~, rw, rtw+F,/R, r , + F , / A , , and A,. In this way, one obtains not only the gas-phase resistances but, concurrently, information about water-phase resistances and the assimilation characteristics of the phycobiont. In general, rtw and r, are confounded with F , and there is apparently no means of determining them individually. However, in our application of the equation to data with R. maciformis we shall be enabled to set r'w=0 under certain conditions, thus providing an estimate of F,. In preliminary experiments with a number of copiously wetted lichens the relationships between A~ and ca did not depart significantly from linearity over a major part of the range of ambient COz concentrations used. This was presumably due to large resistances to CO2 transfer leading to small concentrations of CO2 at the surface of the phycobionts. In terms of the model described by Eq. 5 the condition corresponds to A1 + R
P l a n t a (~) Springer-Verlag 1992
Carbon-dioxide exchange in lichens: determination of transport and carboxylation characteristics I.R. Cowan 1, O.L. Lange*, and T.G.A. Green2 Lehrstuhl ffir Botanik II der Universitfit W/irzburg, Mittlerer Dallenbergweg 64, W-8700 Wiirzburg, Federal Republic of Germany Received 8 October 1991; accepted 11 January 1992
Abstract. Measurements were made o f net rates of CO2 assimilation in lichens at various ambient concentrations o f COs in air and in helox (79% He, 21% 02). Because o f the faster rate o f CO2 diffusion in the pores of lichen thalli when filled with helox than when filled with air, a given net rate of assimilation was achieved at a lower ambient concentration o f CO2 in helox. The differences were used to estimate resistances to diffusion through the gas-filled pore systems in lichens. The technique was first * To whom correspondence should be addressed 1 Permanent address: Plant Environmental Biology Group, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia 2 Permanent address: Department of Biological Sciences, University of Waikato, Hamilton, New Zealand Symbols and units: Rates of assimilation, carboxylation, and respiration are expressed as molar flux of CO2 per mole of chlorophyll (s-1); concentrations as volume COs per unit volume carrier gas (dimensionless); and resistances as concentration differenceper unit rate of assimilation or carboxylation (s).
a = rate of CO2 diffusion in air relative to that in carrier gas (unity if the carrier gas is air and 0.43 if is helox); Al=net rate of COz uptake by the lichen; Ap=gross rate of carboxylation minus photorespiratory decarboxylation in the phycobiont, i.e. net rate of light-activated CO2 exchange; A, = maximum, CO2-saturated magnitude of Ap; c=concentration of CO2; c,=ambient concentration of CO2; ci=c~ minus difference in CO2 concentration across air-filled pore space in the thallus; c~=COz concentration equivalent to partial pressure of CO2 at the surface of the phycobiont; F1 = magnitude of ca at which A1 = 0; F. = magnitude of c. at which Ap = 0; R = rate of "dark respiration" in the lichen (mycobiont and phycobiont); R'= rate of "dark respiration" in region between the surface of the lichen and an arbitrary distance from the surface within the thallus; r = resistance to CO2 transfer from lichen surface to the surface of the phycobiont; rt = resistance to CO2 transfer between effective source of "dark respiration" in the lichen and the surface of the phycobiont; r,, r~=components of r and r*, respectively, attributable to transfer in air-phase; r,~., r~,=comportents of r and r*, respectively, attributable to transfer in waterphase; if=component of r between surface of lichen and an arbitrary distance from the surface within the thallus; r, = resistance to CO 2 transfer and carboxylation in the phycobiont; RH=relative humidity
tested with five lichen species, and then applied in a detailed study with Ramalina maciformis, in which gasphase resistances were determined in samples at four different states of hydration and with two irradiances. By assuming, on the basis o f previous evidence, that the phycobiont in R. maciformis is fully turgid and photosynthetically competent at the smallest hydration imposed (equilibration with vapour at 97 % relative humidity), and that, with this state of hydration, diffusion o f CO2 to the phycobiont takes place through continuously gas-filled pores, it was possible also to determine both the dependence of net rate o f assimilation in the phycobiont on local concentration o f CO2 in the algal layer, and, with the wetter samples, the extents to which diffusion o f CO2 to the phycobiont was impeded by water films. In equilibrium with air of 97 % relative humidity, the thallus water content being 0.5 g per g dry weight, the resistance to CO2 diffusion through the thallus was about twice as large as the resistance to CO2 uptake within the phycobiont. Total resistance to diffusion increased rapidly with increase in hydration. At a water content o f 2 g per g it was about 50 times as great as the resistance to uptake within the phycobiont and more than two-thirds o f it was attributable to impedance of transfer by water. The influences of water content on rate of assimilation at various irradiances are discussed. The analysis shows that the local CO 2 compensation concentration o f the phycobiont in R. maciformis is close to zero, indicating that photorespiratory release o f CO 2 does not take place in the alga, Trebouxia sp., under the conditions of these experiments.
Key words: Carbon dioxide (diffusion and carboxylation resistance) --Helox - Lichen - Photosynthesis (lichen) Water content (lichen)
Introduction Symbiosis in a lichen seems to be an awkward partnership. The initiation and maintenance of metabolic activ-
I.R. Cowan et al. : Carbon-dioxide exchange in lichens ity in phycobiont and mycobiont requires that water be taken up and stored in the lichen thallus, yet the swelling of the fungal hyphae, and the blocking of pores within the matrix of hyphae by extracellular water, consequent on hydration, impedes the diffusion o f COz to the phycobiont (Lange and Tenhunen 1981; Snelgar et al. 1981a; see Kershaw 1985). Our understanding of the ecophysiology of lichens would be m u c h improved if the effects of hydration on metabolism and physical resistance to CO2 diffusion could be analysed individually, but the effects have proven difficult to separate. As water is freely evaporated f r o m the entire surface o f the thallus exposed to the atmosphere, measurement o f v a p o u r loss cannot be used, as it can with leaves o f higher plants, to provide information relevant to the passage of CO2 into the plant tissue. Green and Snelgar (1981) attempted to determine resistances to COz transfer in Stieta lat~'ons and Pseudocyphellaria amphisticta (= P. tividofusca) by analysing the dependence of net rate of assimilation of COz, A1 in each lichen on ambient CO2 concentration ca, having assumed a particular f o r m for the dependence o f net rate o f assimilation in the phycobiont, Ap on the local concentration experienced by the phycobiont % There is some circularity in the logic of this approach, as the f o r m of the function Ap (cs) is a property one wishes to determine without presumption. Later, Snelgar et al. (1981 b) estimated resistances to CO: transfer in the same species using measurements of the a n a t o m y of the cyphellae and pseudocyphellae, and obtained rough agreement with the previous estimates with S. latifrons, but p o o r agreement with P. amphisticta. In this article we describe a new approach to the problem: the use o f a technique previously exploited to study gaseous diffusion processes in animals, and in higher plants (Egorov and K a r p u s h k i n 1988; Parkhurst and M o t t 1990). The relationships A1 (ca) were determined in both air and in helox (79% He, 21% Oz). As CO2 diffuses 2.3 times faster in helox than in air, any given rate of assimilation is achieved with a lower concentration of CO2 in helox than in air. The differences in concentration were used to determine the resistances to gas diffusion in the lichen thalli. We describe b o t h exploratory measurements with five lichen species, and a more comprehensive investigation with Ramalina maciformis at four different water contents and two light intensities. Ramalina maciformis is a particularly appropriate species to use, not only because it has been the subject of m a n y ecophysiological studies (e.g. Lange 1969; Lange et al. 1970; K a p p e n et al. 1979). The green algal phycobiont becomes fully turgid (Btidel and Lange 1991) and appears to be fully competent photosynthetically (Lange et al. 1989) when the lichen is moistened by equilibration with v a p o u r at a relative humidity ( R H ) o f only 97%, equivalent to - 4 0 bar water potential. A t this pressure, only pores having radii less than a b o u t 4 gm are filled with liquid water, and it is reasonable to assume that the thallus contains a continuously gas-filled pore system extending f r o m the external surface to the algal layer. Therefore, the entire resistance to CO2 diffusion (except that in the phycobiont) m a y be estimated by comparison of assimilation characteristics in air and
283 helox, and used to determine how the carboxytation rate in the phycobiont responds to variation in local C O / concentration.
Materials and methods
Material. Preliminary measurements were made with one liverwort and five species of lichens, followed by a more comprehensive investigation with the lichen Ramalina maciformis. The Marchantiales liverwort, Monoclea forsteri Hook., was collected from a native evergreen forest in the Hakarimata Ranges near Ngaruawahia, New Zealand, and kept wet until, and during, the course of the experiments. The foliose lichen species Pseudo~vpheltariafaveolata (Delise) Malme (green phycobiont), P. cotensoi (Church. Bab.) Vainio (green phycobiont), Sticta fuliginosa (Dickson) Ach, (bluegreen phycobiont), S. latifrons A. Rich. (green phycobiont) were collected at Mt. Tarawera near Rotorua (New Zealand), from regenerating forest on the rift plateau. The bushy-fruticose R. maciformis (Del.) Bory (green phycobiont), which is typical of the Negev Desert, was collected near Shivta (Israel). The lichen thalli were transported in air-dry conditions and subsequently stored at low temperatures. Some characteristics of the samples used are given in Table I. Chlorophyll contents were determined according to Ronen and Galun (1984). Apparatus, The equipment, which measures CO2 exchange by samples in each of four cuvettes with controlled COz concentration, temperature and water-vapour pressure, is fully described in Lange and Redon (1983) and Lange et al. (1986). Helox was provided in tanks, the oxygen content of each mixture having been verified with an oxygen electrode. The infra-red absolute and differential COz analysers (Binos, Leybold, Hanau, FRG) were calibrated, with air and helox as the carrier gases, using mixing pumps (Digamix, W6sthoff, Bochum, FRG; see Beyschlag et al. 1986). The sensitivity of the instruments was 5-10% less with helox than with air in both absolute and differential modes of measurement, the difference in the absolute mode increasing with CO2 concentration. During both calibration and measurement, all gas streams entering the IRGA were first brought to uniform concentration of water vapour by humidification at room temperature followed by dehumidification at 2~C in a Peltier-operated vapour trap. In this way errors due to any sensitivity of the analyser to water vapour were avoided. All mass-flow meters and mass-flow controllers (Tylan, Carson, Calif., USA) were also calibrated for air and for helox. Their sensitivity was about 30% greater with helox than with air, corresponding to the difference in the heat capacities per unit volume at constant pressure of the two mixtures. Finally, the accuracy of the complete operational gas-exchange measurement system was tested by use of a small mass-flow controller (Tylan) to inject air or helox into one of the measuring cuvettes to simulate the dilution of CO2 in the Table 1. Species and characteristics of samples
Species
Mass/unit area (g' m -2)
Chlorophyll/ unit area (mmol. m -z)
Monoclea forsteri Pseudocyphellariafaveolata Pseudocyphellaria colensoi Sticta fuliginosa Sticta latifrons Ramalina maciformis Ramalina rnaciformis (O) Ramalina maeiformis (A)
44 75 14I 103 114 264 214 223
0.38 0.28 0.27 (0.31)~ 0.34 0.55 0.28 0.35
For symbols see Fig. 5 " Not known: chlorophyll per unit mass was taken as that in S. tatifrons
284 main gas stream brought about by assimilation in a lichen sample. The system estimated apparent rate of assimilation accurately when CO2-free air was added to air streams having various CO2 contents, but underestimated it by 3 % when pure helox was added to streams of helox having various CO2 contents. Allowance was made in all experiments for this discrepancy, the cause of which was not identified. In order to estimate the resistance to CO2 transfer of the boundary layer between the ambient gas and the lichen surfaces, the rate of evaporation from a wet filter paper having approximately the area of the samples to be studied (30 cm2), placed in one of the cuvettes, was measured. The boundary-layer resistance to vapour transfer per unit area was found by applying energy-balance theory to calculate the temperature of the paper (Cowan and Farquhar 1977), and was less than 2 m 2 9s 9mol-x, equivalent to a resistance to CO2 transfer of 3 m 2 9 s 9m o l - t. The smallest resistances to CO2 transfer per unit area within and from lichens during photosynthesis were estimated to be 30 m z 9s 9m o l - ~ and therefore the characteristics of air flow in the cuvettes can have had little effect on CO2 assimilation rates and CO2 concentrations in the lichens. Figure 2 shows measurements of assimilation rates in a sample of the liverwort, M. forsteri, having about the same capacity for carbon fixation as the lichen samples but, unlike lichens, containing no internal airspace. That the assimilation characteristics in air and in helox were identical indicates that the resistance of the boundary layer was indeed negligible.
Experimental procedure. The lichens used in the first experiment were activated by wetting the thaUi and keeping them at 10~ C with a 12:12 h light:dark cycle for 4 d before commencing measurements. They were then rewetted by spraying with liquid water, the excess water being removed by blotting the undersides of the thalli with tissue paper (Snelgar 1981), and placed in the cuvettes. The two samples of R. maeiformis used in the more detailed experiment were initially activated by humidification at 97 % RH in the experimental cuvettes for 56 h. Following measurements of CO2 exchange at this state of hydration, further measurements were made with one of the samples having been wetted by immersion and the other having been infiltrated with water under vacuum (water-jet pump). A third set of measurements was made with the water content of the first sample reduced by blotting and then increased by light spraying, and that of the second sample maintained by re-infiltration. The water contents corresponding to the four conditions of hydration, vapour-equilibrated, lightly wetted, copiously wetted and infiltrated, were about 0.5, 1.2, 2 and 6 g per g dry weight, respectively. Water loss from the illuminated lichen samples during the experiments was unavoidable, but was minimized by supplying air of 97 % RH to the cuvettes. Rate of assimilation was especially sensitive to changes in water content of the thalli when in quasi-equilibrium at this humidity, and therefore each measurement in the light (which took 20 min) was followed by a period of 3 to 4 h darkness in which loss of water was made good by vapour re-equilibration. Moistening of the lightly wetted sample was repeated in the course of measurements and the small changes in water content brought about may have been responsible for some scatter in the data obtained. Measurements of net rate of assimilation at various ambient concentrations of CO2 in air and in helox were made with each lichen (Fig. 2), and, with R. maeiformis, in each state of hydration (Fig. 3). Photosynthetically active irradiance was 120 lamol- m - 2 - s - t in the preliminary experiments, except with R. maeiformis which received 300 ~tmol 9m - 2. s- 1. These irradiances were sufficient to induce maximum rates of photosynthesis with ambient concentrations of CO2 in the range 0 to about 300 lal 91-1. The more detailed measurements with R. maciformis were carried out with two irradiances: 105 and 300 lamol' m -2" s -x. Cuvette temperature was 15.0 4- 0.1 o C in all experiments. Concentrations of CO2 were step-changed in the range 0 to about 350 ~tl - 1-t with some measurements (data not shown) being made at concentrations up to 2,500 ~tl 91-1. During the course of each set of measurements, frequent switches were made between air
I.R. Cowan et al.: Carbon-dioxide exchange in lichens and helox as the carrier gas. Following each change of C O 2 c o n and-or carrier gas, an interval of at least 15 rain was allowed to elapse before the subsequent measurement was made to ensure that a steady rate of assimilation had been achieved. The completion of a set of measurements was most protracted (72 h) with R. maciformis in the vapour-equilibrated state. Measurements of rate of respiration in the dark were made for each lichen sample and each state of hydration. Rates in air and in helox were identical, as were the net rates of assimilation at a given irradiance with a saturating concentration of CO2. Therefore we concluded that the substitution of helox for air did not directly affect either respiratory or photosynthetic metabolism of the lichens and that all effects caused by change of carrier gas could be attributed to differences in the speeds of CO2 diffusion in the lichen thalli. centration
Theory Our experiments had three interrelated objectives: to d e t e r m i n e r e s i s t a n c e s to C O 2 t r a n s f e r in l i c h e n thalli, e s p e c i a l l y as a f f e c t e d b y w a t e r c o n t e n t ; to e s t i m a t e l o c a l c o n c e n t r a t i o n s o f C O 2 in t h e v i c i n i t y o f t h e a l g a l p h y c o b i o n t s ; a n d to e x a m i n e t h e d e p e n d e n c e o f C O 2 f i x a t i o n r a t e s in t h e p h y c o b i o n t s o n l o c a l c o n c e n t r a t i o n o f C O 2 . I n Fig. 1a t h e f l o w p a t h o f C O 2 f r o m a m b i e n t a t m o s p h e r e , in w h i c h t h e c o n c e n t r a t i o n is ca, to t h e s u r f a c e o f t h e p h y c o b i o n t in t h e a l g a l layer, w h e r e t h e c o n c e n t r a t i o n is % is r e p r e s e n t e d b y a single resistor. I f t h e n e t r a t e o f C O 2 u p t a k e b y t h e l i c h e n is A1 a n d t h e n e t r a t e o f d a r k r e s p i r a t i o n in b o t h m y c o b i o n t a n d p h y c o b i o n t is R , t h e n A t + R = Ap, say, is t h e g r o s s r a t e o f c a r b o x y l a t i o n m i n u s p h o t o r e s p i r a t i o n in t h e p h y c o b i o n t . I n g e n e r a l , d a r k r e s p i r a t i o n is r e l e a s e d at v a r y i n g r a t e s a l o n g t h e e n t i r e f l o w p a t h , b u t it is c o n v e n i e n t to d e f i n e a single p o i n t at
a A~
AI*_~R'
Ap
s-O I
r'
r - r'
i t I I
t I t
R'
AIj,.
i
R-R'
I
A I+._.R
._~,..: AI +R
/
rg - rg*
rw - rw* I
rg~
rw'
/R Fig. la, b Scheme of fluxes and resistances used to describe diffusion of CO2 in a lichen from external atmosphere (left) to phycobiont (right). In a, the catena of resistances is arbitrarily divided in two, the resistance components being r' and r-r'. The flux at the dividing point in the catena &resistances is intermediate between the net rate of assimilation of the lichen (At) and that of the phycobiont tAp) being the sum of At and the rate of respiration (R') in the portion of thallus external to that point. In b, the catena is split in two at the effective centre of respiration rate (R) in the thallus and further subdivided into gas-phase and water-phase components, distinguished by subscripts g and w
I.R. Cowan et al.: Carbon-dioxideexchangein lichens
285
which it is effectively all evolved. Let the rate of respiration released into a segment of the flow path, resistance r', extending from the surface to any arbitrary distance in the thallus be R'. Then A~ + R' = - d c / d r ' , and by integration over the whole path, resistance r, from surface of the lichen to the surface of the phycobiont it follows that ca = A l r + R r t + c ~
(Eq. 1)
Ap= AI+R-
where 1 /'r r* = ~ [ R ' d r ' J 0
The resistance rt has a maximum of r, which would obtain if all of the respiration took place at the surface of the thallus, and a minimum of zero, which would obtain if all of the respiration took place within, or at the surface of the phycobiont. If respiratory COz were evolved uniformly along the resistance path, for example, then R ' = r'R/r, leading to r* = r/2. In general, r* defines the position of the "centre of gravity", as it were, of dark respiration. Therefore the system can now be represented by Fig. lb, with the resistances in the gas-phase and water-phase components of the flow path being distinguished by subscripts g and w respectively. The gas-phase in the lichen normally consists of air, of course. If the lichen is placed in helox, account must be taken of the change in diffusion rate of CO2. In general, the relationship between CO2 transport and concentration difference is described by the equation ca = A1 (err,+ rw) + R (ctrtg+r~) + cs
(gq. 2)
in which a is unity with air and 0.43 with helox. As the carbon metabolisms of mycobiont and phycobiont are not affected by the substitution of helium for nitrogen, the concentration c, corresponding to any particular net rate of assimilation, A1, attained with a particular set of environmental conditions will be independent of the carrier gas used. However, the ambient concentration of COz required to achieve that rate of assimilation will be greater with air than with helox because of the smaller diffusivity of CO2 in air. From Eq. 2, the difference is
kca = 0.57" (A~rg+ Rrgt)
helox to give exactly the same net rates of assimiliation. Interpolation is required. It is desirable that the mathematical function used be consistent, not only with the model in Fig. lb, but also with a plausible description of the assimilation characteristics of the phycobiont. We express the latter in the form of a Michaelis-Menten response to CO2 concentration with allowance made for photorespiration, i.e.
(Eq. 3),
the numerial factor being the difference between a for air and for helox. Thus Aca is a linear function of A1 for constant conditions of thallus water content, temperature, and irradiance. The resistance rg is defined by the slope, and, provided R may be equated with rate of respiration measured in the dark t, the resistance rg* is defined by the intercept at A1 = 0. In practice, however, Ac, is not found directly, as it is inconvenient to adjust concentrations of CO2 in air and It seemsunlikelythat respiration in the mycobiontis substantially suppressed in the light(see Kappenand Lange 1972). Recentstudies with a higher plant (McCashin et al. 1988) and with algae (Weger and Turpin 1989; Turpin 1991)indicate that dark respiration in the phycobiont may also be expected to continue essentiallyundiminished in the light, at least over the range of illuminationused in the present experiments.
cs- F , r, + c J A ,
(Eq. 4)
where F , is the magnitude of c~ at which Ap = 0 (i.e. at which rate of gross carboxylation exactly balances rate ofphotorespiration), r, is the resistance to C02 transport and carboxylation in the phycobiont, and A , is the maximum, C02-saturated magnitude of Ap. Using this equation to eliminate cs in Eq. 2, it follows that ca = Al(~rg+rw)+R (arg+ * rw+ * F./R) +
(A1 + R) (r, § F , / A , )
1 -- (At + R ) / A ,
(Eq. 5)
This is a quadratic equation in At and is readily inverted to express A1 as a function of the two quantities, Ca and a, that are varied in each experiment - the water content, temperature, and irradiance of the lichens being constant. It may be fitted to the data, using an algorithm minimising the mean square deviation in A1. If R is known the process yields estimates of six parameters: rg, r~, rw, rtw+F,/R, r , + F , / A , , and A,. In this way, one obtains not only the gas-phase resistances but, concurrently, information about water-phase resistances and the assimilation characteristics of the phycobiont. In general, rtw and r, are confounded with F , and there is apparently no means of determining them individually. However, in our application of the equation to data with R. maciformis we shall be enabled to set r'w=0 under certain conditions, thus providing an estimate of F,. In preliminary experiments with a number of copiously wetted lichens the relationships between A~ and ca did not depart significantly from linearity over a major part of the range of ambient COz concentrations used. This was presumably due to large resistances to CO2 transfer leading to small concentrations of CO2 at the surface of the phycobionts. In terms of the model described by Eq. 5 the condition corresponds to A1 + R
