Plant responses to high O2 concentrations: relevance to previous high O2 episodes

Palaeogeography, Palaeoclimatology, Palaeoecology (Global and Planetary Change Section), 97 (1991) 19-38

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Elsevier Science Publishers B.V., Amsterdam

Plant responses to high

concentrations: relevance to previous high O 2 episodes 0 2

John A. Raven Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK (Received October 20, 1990; accepted April 10, 1991)

ABSTRACT Raven, J.A., 1991. Plant responses to high 0 2 concentrations: relevance to previous high 0 2 episodes. Palaeogeogr., Palaeoclimatol., Palaeoecol. (Global Planet. Change Sect.), 97: 19-38. Exposure to the ~ 43 kPa O z thought to correspond to the highest 0 2 partial pressure in the Phanerozoic rather than the 21 kPa in the present atmosphere deleteriously affects extant C 3 land plants by increasing (1) the O 2 inhibition (competitive with CO z) of the carboxylating enzyme ribulose 1,5 bisphosphate carboxylase-oxygenase, (2) the 0 2 inactivation of nitrogenase in symbiotic N2-fixers, and (3) the generation of reactive oxygen species which can damage nucleic acids, proteins, lipids and other cell constituents. Exposure of these plants to 0 2 levels approximating a high 0 2 episode inhibits growth via inhibition of ribulose bisphosphate carboxylase and, possibly, by unrepaired damage caused by reactive oxygen species and by the resource diversion related to increasing the capacity of mechanisms reducing the level of reactive oxygen species and to repairing the damage caused by these species. Longer term effects (e.g. increased level of mutations in a population) have not been explored. Natural exposure of all or part of extant plants to 0 2 levels in excess of 43 kPa occurs in the light in most plants other than C 3 land plants due to photosynthetic 0 2 evolution with restricted diffusion to a medium with normal 0 2 levels attendant on the maintenance of high CO 2 concentrations around ribulose 1,5 bisphosphate carboxylase. While the high 0 2 levels seem to be restricted where possible to non-meristematic regions of multicellular plants, these plants which have high internal 0 2 levels do not seem to be disadvantaged relative to C 3 land plants. The high O 2 concentrations proposed for periods in the Pbanerozoic are tolerated, as intracellular 0 2 levels, in some cells of a substantial fraction of extant phototrophs, and many can tolerate substantially higher levels. Fire seems more likely than the effects discussed in this paper as limitations on co-occurrence of terrestrial vegetation and high 0 2 levels.

Introduction Geochemical evidence suggests that considerable variations in atmospheric O 2 mass have occurred during the change from the Hadean, essentially O2-free atmosphere to the present atmosphere containing some 38.1018 mol 0 2 (cf. Budyko et al., 1987; Berner and Canfield, 1989). Variations in the mass of 0 2 in the Phanerozoic atmosphere between 8.1018 and 92.10 ~8 tool 02, with three main peaks and three troughs, have been suggested by Budyko et al. (1987). Using a more acceptable methodology, Berner and Canfield (1989) have computed Phanerozoic O 2 variations between 23.1018 and 78.1018 mol 02, with

only two main peaks and troughs. Translated into sea-level partial pressures of O 2, the Berner and Canfield (1989) variations are from 12.7 kPa to 50.8 kPa, as compared to the extant value of 21 kPa. Cells of individual extant organisms are naturally exposed to O 2 concentrations in solution which correspond, in an equilibrium gas phase, to a range of O 2 partial pressures substantially greater than the range proposed for Phanerozoic atmospheres. O 2 is not subject to active transport by organisms, i.e. 0 2 is not moved by energy-requiring catalysts against an 0 2 free energy gradient. This means that, no matter how effective any facilitated transport processes are (gills, lungs,

0921-8181/91/$03.50 © 1991 - Elsevier Science Publishers B.V. All rights reserved

2(]

blood circulation, etc.), the 02 concentration in respiring cells in the steady-state is lower than that in the immediate environment of the organism. Hypoxia, and temporary or permanent anoxia, are thus common fates for many organisms, or parts of organisms, in nature (Hochachka, 1980; Raven, 1984a; Crawford, 1987). Many organisms are thus able to survive, and in many cases grow, at intracellular 0 2 levels lower than those corresponding to air-equilibrium at the suggested O 2 minima during the Phanerozoic, although it is clear that their O: supply problems would have been exacerbated during these 0 2 minima, with the likelihood of greater prevalence of anoxic sediments, restrictions on diving by air-breathing aquatic animals, etc. Problems of hypoxia and facultative or obligatory capacity to live in anoxic conditions, have been relatively well investigated, and the emphasis of this paper is on hyperoxia, both natural and experimental, in extant organisms as a possible indication of the responses of organisms to Phanerozoic high-O 2 episodes. The discussion will mainly relate to plant responses, although we note that the 12.7 kPa 0 2 trough of Berner and Canfield (1989) corresponds roughly to an altitude of 4000 m and that many people live close to, or even higher than. this altitude (Gilbert, 1981).

Occurrence of hyperoxia in extant organisms An 0 2 concentration inside the cells which exceeds the normal value can be induced in organisms by increasing the external 0 2 partial pressure or dissolved 0 2 concentration for terrestrial and aquatic organisms respectively. Such experimental studies are generally of short duration, and tell us little about long-term effects of hyperoxia on the organisms, such as the extent to which any inhibitory effects noted in short term investigations can be ameliorated by genetic change in the long term. Induced or 'spontaneous' mutations, or the production of transgenic organisms, which have altered responses to hyperoxia, do not necessarily reflect the capacity for acclimation to hyperoxia which could be achieved in a genetically variable population undergoing natural selection.

J.A. RAVEN

Examples of natural exposure of part or all of organisms to hyperoxia should give a better indication of the potential for acclimation, although in cases in which the hyperoxia involves accumulation of 0 2 within compartment(s) in the organism the possibilities for imposing well-characterised changes in intracellular 0 2 concentration may be more limited than is the case for organisms lacking such compartments with their attendant 0 2 diffusion barriers. However, such examples of natural hyperoxia arc important in interpreting the acclimatory capacities of cells and organisms, and we set the scene by considering the organisms in which natural hyperoxia is experienced, and the extent of hyperoxia for organisms growing in (or in an aqueous medium in contact with) air. It must be emphasized that in the absence of active 0 2 transport (see above), natural hyperoxia in photolithotrophs only results from the occurrence of photosynthetic 0 2 evolution under conditions in which equilibration of O 2 between the sites of 0 2 production and a bulk medium of air, on air-equilibrated water, is restricted (cf. the high 0 2 partial pressures in the swim bladders and analagous organs in aquatic metazoa: see Gilbert, 1981). A seminal paper in this area is that of Samish (1975). He points out that plants which are supplied with CO 2 by diffusion from the present-day atmosphere and which use ribulose bisphosphate carboxylase-oxygenase (RUBISCO) as their initial carbox3'lase, i.e. C 3 plants, in which a 3 carbon compound (phosphoglyceric acid) is the first stable product of CO 2 fixation, have a very limited O 2 build-up in their photosynthetic tissues. This conclusion is based on the observations that CO 2 entry and 02 loss use the same path during photosynthesis and that as much CO 2 moves into the leaf as 0 2 moved out of the leaf in a given time. Fick's law then indicates that the overall gradients between bulk phase and site of photosynthesis are similar for CO 2 and for O 2, with a rather low gradient for O: resulting from its higher diffusivity in both the gaseous and the aqueous portions of the pathway. The maximum gradient for CO 2, expressed as dissolved CO2, cannot exceed 10 mmol m 3 at 25°C if the plant is to carry out net photosynthesis, since the air-

P L A N T R E S P O N S E S q'O H I G H ()~ C O N C E N T R A T I O N S

equilibrium CO 2 concentration is only 11.9 mmol m 3 and C 3 plants have a finite CO x compensation concentration below which they cannot lower the CO 2 concentration; this has a value of at least 1 mmol m 3 at 25°C in the presence of air-equilibrium 0 2 concentrations (Raven, 1984a). Accordingly, the maximum gradient for 0 2 also cannot exceed 10 mmol m 3. Since the air-equilibrium O, concentration at 25°C is 236 mmol m -~, the O, concentration in cells of illuminated leaves will not exceed 246 mmol m -3. Even granted facilitation of aqueous-phase movement of C O 2, but not that of 0 2, by carbonic anhydrase (Samish, 1975; Raven, 1977a; Raven and Glidewell, 1981; Cowan, 1986), the 0 2 concentration in photosynthesising cells is unlikely to exceed 260 mmol m 3 i.e. 1.1 time the air-equilibrium concentration (see Table 1). C 3 plant species constitute 80-90% of the extant terrestrial flora, and were even more dominant earlier in the Phanerozoic. The other two major categories of higher plants with respect to CO, assimilation, the C 4 plants (i.e. those in which a 4 carbon compound (oxaloacetie acid) is the first stable product of CO 2 fixation in the light) and Crassulacean Acid Metabolism (CAM) plants, both show substantial computed (C 4) or measured (CAM) O 2 build-up in their photosynthetic tissues (Table 1). The reasoning underlying the computations on the C 4 plants relates to the restricted gas transfer between the cells to which the C~-C 4 organic acid cycle 'pumps' CO 2 (originally fixed by phosphoenolpyruvate carboxylase) and where refixation by RUBISCO can occur, and the gas phase. This restricted gas transfer greatly reduces CO 2 leakage from the compartment in which RUBISCO functions, and thereby increases the energetic, nitrogen and water efficiency of C 4 photosynthesis (Edwards and Walker, 1983). However, restricting CO 2 leakage also restricts 0 2 leakage, thus increasing the steady-state 0 2 concentration in the cells where RUBISCO occurs in those cases [the NADme (NAD malic enzyme) and PEPck (phosphoenolpyruvate carboxylase) subtypes] in which 0 2 evolution occurs associated with RUBISCO activity (Raven, 1977a; Jenkins et al., 1989): see Table 1. The ostensible 'use' of the

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CO 2 pump is improving the working conditions of RUBISCO which involves increasing the [CO2]:[O2]ratio around RUBISCO, and the high steady-state O 2 concentration can be construed as counteracting the CO2, pump. However, the [CO2]:[O2] is maintained high enough not to prejudice the impact of the CO 2 pump on RUBISCO (Raven, 1977a; Jenkins et al., 1989). In contrast to the C 4 plants where the high intracellular 0 2 concentration is indirectly computed, the high 02 concentration in green tissues of CAM plants during the light deacidification phase, where gas exchange between the photosynthetic tissues and the atmosphere is greatly restricted, has been directly measured (Table 1). As with C 4 plants, the high O~ level around RUBISCO is offset, as far as the carboxylase: oxygenase competition is concerned, by high CO2 levels during the deacidification phase in CAM plants (Spalding et al., 1979; Griffiths, 1988). The other main, if heterogeneous, group of organisms which have higher than atmospheric equilibrium intracellular O 2 concentrations during photosynthesis are a number of aquatic 0 2 evolvers, including algae, aquatic higher plants, and symbioses of algae with a range of invertebrates (Table 1). The general feature of photosynthesis which permits these high steady-state O, concentrations is the presence outside the cells of high inorganic C concentrations. In some freshwaters, the CO 2 concentration is greatly in excess of atmospheric equilibrium thanks to CO 2 enrichment from the soil solution dominated by heterotrophic activity. Here CO 2 supply to RUBISCO is possibly down a much greater CO~ concentration difference than is the case for terrestrial plants, with a corresponding possibility of a larger 0 2 concentration difference from inside to outside. However, an intracellular steady-state 0 2 concentration much in excess of air-equilibrium is unlikely, since soil respiration which augments the CO 2 in groundwater decreases the 0 2 in, theoretically, a ratio of near 1 : 1. The means whereby the aquatic phototrophs attain intracellular 0 2 concentrations in excess of air-equilibrium relies on active movement of inorganic C from the medium to the intracellular phase. This active transport frequently involves

2 3-

J.A RAVEN

TABLE 1 Hyperoxia inside and outside a number of O2-evolving phototrophs Organisms, environment

Mechanism of inorganic C acquisition

Intracellular (i) or extracellular (e) concentration of 0 2 according to computation (c) or measurement (m)

(1) C, Terrestrial plants

CO 2 diffusion from the atmosphere to RUBISCO

236-250 mmol m 3 (i) (c) (1)

(2) C 4 terrestrial flowering plants

CO 2

diffusion from the atmosphere to PEPc, with movement of the product within the tissue acting as a CO, pump into the compartment containing RUBISCO

236-250 mmol m 3 (i) (c) in NADPme subtype (2, 3)

CO 2 diffusion from the atmosphere to PEPc at night, storage of fixation products in the vacuole, re-release of CO z in day with re-fixation by RUBISCO

890

CO 2 maintained at higher concentration around RUBISCO than in an air-equilibrium solution

418 mmol m 3 (i) (m) in siphoneal blood of the giant clam Tridacna symbiotic with Symbiodinium (6)

(3) CAM terrestrial plants and ferns

flowering

(4) Aquatic phototrophs with inorganic C concentrating mechanisms

40-550 mmol m 3 (i) (c) in NADme and PEPck subtypes (2, 3) mmol

m 3 (i)

(m)

(4)

474 mmol m 3 (i) (m) (5)

389 mmol m -3 (i) (m) in gastrodermal tissue of the coelenterate Anthopleura symbiotic with Sym-

biodinium 570 mmol m 3 (e) (mt at surface of Globigerinoides symbiotic with Symbiodinium (7) (4) Aquatic phototrophs with inorganic C concentrating mechanisms

CO 2 maintained at higher concentration around RUBISCO than in an air-equilibrium solution

920 mmol m -~ (e) (m) at surface of brown seaweed Scytosiphon, 750 mmol m 3 (e) (m) among epiphytes (8) 600 mmol m 3 (e) (m) at surface of seagrass Zostera, 650 mmol m 3 (e) (m) among epiphytes (8) 650 mmol m ~ (e) (m) at surface of flesh-water macrophyte Potamogeton, 780 mmol m -3 (e) (m) among epiphytes (8) 700 mmol m 3 (e) (m) in rock pool containing seaweeds (9) 1150 mmol m -3 (e) (m) at 1 mm depth in marine sediment dominated by pennate diatoms (10) 700 mmol m 3 ( e ) ( m ) i n a solution in equilibrium with a gas phase in the coral Millepora (11)

References cited by number in column 3 are: (1) Samish (1975), (2) Raven (1977a), (3) Jenkins et al. (1989), (4) Ekern (1965), (5) Spalding et al. (1979), (6) Dykens and Shick (1982), (7) Jorgensen et al. (1985), (8) Sand-Jensen et al. (1985), (9) Ganning (1971), (10) Revsbech et al. (1981), (11) Bellamy and Risk (1982).

P L A N T R E S P O N S E S T O H I G H ()~ C O N C E N I ' R A T I O N S

HCO 3 but can involve C02, and in marine organisms is often related to the 2 tool m -3 H C O f in seawater (Raven, 1984a). The mechanism of 0 2 accumulation is analogous to that in C 4 and CAM plants: 0 2 is produced in a compartment which is supplied with CO 2 other than by simultaneous CO z diffusion from the bulk medium, and with restricted diffusive exchange of CO 2 and 0 2 between the bulk medium and the compartment in which RUBISCO acts (Table 1). In some cases (e.g. the cyanobacterium Synechococcus) the computed intracellular concentration of O, during steady-state photosynthesis reaches very high levels in the original model of inorganic C behaviour in the cell (Badger et al., 1985). However, subsequent models permit substantially lower steady-state intracellular O z concentrations (Reinhold et al, 1987; Price and Badger, 1989a,b). Another aspect of aquatic phototrophs in restricted volumes of water with high inorganic C concentrations is that, with limited 0 2 exchange between the water and the atmosphere, extracellular 0 2 can accumulate to high levels. This occurs in many high intertidal rock pools and small bodies of freshwater (Table 1). The data in Table l show that a number of major categories of phototrophs are exposed to O, concentrations of twice air.equilibrium values or more in at least some parts of their photosynthesising tissues. In terms of the number of described species involved the C 3 terrestrial plants, in which 0 2 accumulation within the tissues is minimal, outnumber the other 0 2- evolving phototrophs in which 0 2 accumulation occurs to a greater extent. However, in terms of global productivity, and of the biomass of O2-evolving tissues involved, the organisms with substantial accumulation of 0 2 in photosynthesising tissues are likely to be more nearly equal to the terrestrial C 3 plants (Griffiths, 1988; Johnston and Raven, 1989; Raven and Johnston, 1991). Effects of hyperoxia on extant phototrophs

Background As noted in the Introduction, the influence of hyperoxia on plants can involve competitive inhi-

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bition of the carboxylase activity of RUBISCO and stimulation of its oxygenase activity while, in N2-fixing symbioses, it can involve increased inactivation of the nitrogenase enzyme complex. These two effects are additional to the general effects of hyperoxia in all organisms regardless of their capacity to i.e. an photosynthesise, increase in the rate of production of toxic oxygen species with increased 02 concentration (Halliwell and Gutteridge, 1989).

Influence of hyperoxia on growth i,ia effects on photosynthesis Many short-term experiments have shown that the rate of net photosynthesis in terrestrial C 3 plants is decreased if the 0 2 partial pressure is increased above extant levels while the CO 2 partial pressure is maintained at its present value (Edwards and Walker, 1983). Fewer data are available for the long-term effects of hyperoxia on photosynthesis and on growth (Quebedaux and Hardy, 1975), but it is clear that the effects are essentially the same as those found in the short-term investigations for terrestrial C plants (Quebedaux and Chollet, 1977), i.e. no acclimation is evident. Although not directly related to photosynthesis, it is of interest that reproductive effort, expressed as the fraction of plant dry mass allocated to seeds, is essentially unaltered when Glycine max is grown at 40 kPa rather than 21 kPa 0 2, while 5 kPa 0 2 substantially reduces reproductive effort, when this C 3 plant is growing at extant atmospheric CO 2 levels (Quebedaux and Hardy, 1975). Dry matter production decreases in the order 5 kPa 0 2 > 21 kPa 0 2 > 411 kPa 0 2, as expected from RUBISCO kinetics at limiting CO 2 partial pressures (Quebedaux and Hardy, 1975). By contrast to the effects on C 3 terrestrial plants, increase in 0 2 to twice the extant atmospheric partial pressure or its equivalent in solution, while maintaining CO 2 at the extant atmospheric value or its equivalent in solution, has little effect on growth or long or short-term photosynthesis by C 4 terrestrial plants or by aquatics with CO 2 concentrating mechanisms (Quebedaux and Chollet, 1977; Pruder and Bolton, 1979, 1980;

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Edwards and Walker, 1983; Raven, 1984a cf. Pefiuelas, 1987). We have already seen that the build-up of 0 2 within the RUBISCO-containing cells when these plants are photosynthesising at air levels of 0 2 does not inhibit the carboxylase activity or stimulate the oxygenase activity of RUBISCO to any substantial extent: the high CO 2 level in the compartment containing RUBISCO offsets the O, effect. This suppression of the 0 2 effect by the high CO 2 level in the RUBISCOcontaining cells still occurs when the external 0 2 concentration is above atmospheric (or atmospheric-equilibrium) levels. This is relevant to extant habitats such as rock-pools or sediments with restricted diffusive access to a bulk phase with atmospheric 0 2 (or its solution equivalent) levels (see Table 1) as well as to past episodes of high 0 2. The inhibition of photosynthesis (and growth) of terrestrial C 3 plants at atmospheric CO 2 levels due to increased O z concentrations, and the absence of inhibition in plants with CO 2 concentrating mechanisms, implicitly applied to plants receiving optimal supplies of other resources such as photons, water, nitrogen, iron and manganese. Experimental data and theoretical considerations show that C~ plants respond to changed CO:: 0 2 ratios by increased photosynthesis and growth per unit photons absorbed or water transpired, or rate of photosynthesis and growth per unit nitrogen, iron or manganese in the organism (Edwards and Walker, 1983; Raven, 1990) when the CO2: O, ratio is increased relative to the extant atmospheric level. Conversely, a decreased COe: 0 2 ratio leads to a decrease in the resource use efficiency. While the data and computations generally relate to 0 2 levels at or below extant values in combination with a range of CO 2 levels above and below extant values, the conclusions can be very reasonably extended to hyperoxic conditions. The organisms with CO x concentrating mechanisms, by contrast, have been shown (or are likely) to have little or no effect of CO:: O z ratio on resource (photons, water, nitrogen, iron and manganese) use efficiencies, at least with 0 2 levels of up to twice the extant atmospheric levels. These considerations suggest that the competitive advantages of organisms with CO2-concentrating

J.A. RAVEN

mechanisms relative to C 3 plants would be increased at decreased C O 2 : 0 2 ratios both when other sources were in adequate supplies and under a range of resource-limited conditions. The relevance of this to past 0 2 and CO 2 levels, and to timing of the evolution of CO, concentrating mechanisms, will be considered later.

Influence of hyperoxia on growth via effects on nitrogen fixation Two important differences between the effects of hyperoxia on CO 2 fixation by RUBISCO and on N 2 fixation may be noted. One is that the 0 2 effect on nitrogenase is non-competitive and, at the level of individual enzyme molecules, irreversible; restoration of activity requires further protein synthesis. The other is that, as with photosynthesis, nitrogen fixation by eukaryotes is a result of symbiosis with prokaryotes; however, while prokaryotic phototrophs have been fully integrated into eukaryotes, as plastids, producing photosynthetic eukaryotes which originated more than 1 Ga ago, the N2-fixing symbionts are acquired anew from a free-living population in each succeeding generation of the eukaryotes (Sprent and Sprent, 1990). The analogy for the N2-fixing symbioses among the eukaryotic phototrophs are the lichens and the alga-invertebrate symbioses (see Table 1) where the phototrophic partner, pro - - o r eukaryotic, is re-acquired with each generation of the non-photosynthetic component. The predominant means of avoiding 0 2 inactivation of nitrogenase in diazotrophic symbioses is by means of restriction on the entry of 0 2 to the site of nitrogenase. Such restrictions must permit N 2 entry to act as a substrate for nitrogenase, and sufficient 0 2 entry for ATP generation, again as a substrate for nitrogenase; this is not easy in view of the physicochemical similarities of N 2 and 0 2, and the absence of active transport of N 2 (cf. the absence of active transport of 0 2 mentioned above). Of the three taxa of prokaryotes involved in N2-fixing symbioses with eukaryotes, the cyanobacteria and the actinomycete Frankia restrict 0 2 access to nitrogenase by means of lipid layers outside the cytoplasmic membrane of the N2-fixing cells in a filament also containing non-

PLANT RESPONSES TO HIGH 02 CONCENTRATIONS

25

fixing cells, while the rhizobial symbioses have an aqueous barrier surrounding the zone containing the N2-fixing prokaryotic cells. In essence, the first two cases involve a prokaryote-based mechanism, (albeit sometimes with supplementation by eukaryote barrier in some Frankia symbioses) while the third involves a eukaryote mechanism (Sprent and Sprent, 1990). Responses to hyperoxia by these symbioses involve increased numbers of layers of lipid in the case of cyanobacteria and Frankia and, probably, an increased thickness of the aqueous barrier in the rhizobial symbioses (Silvester et al, 1988a,b; Dakota and Atkins, 1990a,b; Parsons and Day, 1990; Sprent and Sprent 1990). At least in the case of the rhizobial symbiosis the imposition of a greater O~ barrier in Vigna unguiculata (L.) Walp. grown with their roots in 40 kPa 0 2 rather than 20 kPa 0 2 causes very little change in N 2 fixation on a plant or a nodule dry weight basis, or in the quantity of respiratory substrate converted to CO 2 per unit N, fixed (plus H 2 unavoidably evolved). Very substantial inhibition of N 2 fixation on a plant or nodule dry, weight basis, and a decrease in the energetic efficiency (increased conversion of respiratory substrate to CO 2 per unit N 2 fixed

and H 2 evolved), occurs at 80 kPa 0 2 around the roots (Dakora and Atkins, 1990a,b). Plant growth is also substantially inhibited with 80 kPa around the N2-fixing roots (albeit with air levels of 0 2 around the shoots); Dakora and Atkins (1990a,b). Symbiotic N 2 fixation would thus accommodate, at the phenotypic (acclimation) level, to a doubling of the atmospheric O, content without a significant decrease in the rate of N, fixation or the energetic efficiency of N 2 fixation. We note that the N2-fixing symbioses involving photosynthetic eukaryotes and cyanobacteria do not involve substantial photosynthetic CO, fixation by the cyanobacteria, even when the cyanobacteria are exposed to light (Sprent and Sprent, 1990). Accordingly, they do not suffer exposure to supra-atmospheric O 2 concentrations as a result of photosynthesis using a CO~ concentrating mechanism. The evidence discussed above suggests, in the context of the likely timing and characteristics of the evolution of symbiotic N 2 fixation (Sprent and Raven, 1986, 1991; Raven and Sprent, 1989) that 0 2 variations of the magnitude suggested by Berner and Canfield (1989) were not major constraints on the evolution of symbiotic N~ fixation.

TABLE 2 Reactive oxygen species in living organisms: means of production and disposal, and the damage whk:h they cause (see Halliwell and Gutteridge, 1989: Raven, 1991) Reactive oxygen species

Means of production

Means of disposal

Main damage caused if not disposed off

H 2° 2 (hydrogen peroxide)

Essential redox processes in presence of O~

Catalase (produces H 2 0 and 1/202) Peroxidases and reductant co-substrate (produces H202 and oxidized substrate)

Production of •OH

O, (superoxide radical ion)

Essential redox processes in presence of O,

Superoxide dismutases and H + (produces 1/2H202 + 1/202)

Production o f . OH

-OH (hydroxyl radical)

From H 2 0 > 0 2 aa-toeopherol

Scavengers such as acids, proteins,

Reacts with nucleic lipids, etc.

IO~ (singlet oxygen)

From 02 and photoproduced excited states (e.g. in photosynthesis)

Quenchers such as /~-carotene

Reacts with many compounds

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Hyperoxia and the production of reactiue oxygen species

The production of reactive oxygen species involves interaction of 0 2 with redox intermediates of photosynthesis, respiration, and microbody processes to yield O: and H 2 0 2 and, by subsequent reactions, to the highly toxic hydroxyl radical, •OH, and with photoproduced excited states to produce the very reactive singlet oxygen, O 2 (Halliwell and Gutteridge, 1989): see Table 2. While the rate of production of these toxic products is increased by hyperoxia, the increase is not necessarily in direct proportion to the increase in O z concentration (Halliwell and Gutteridge, 1989). The damage due to these reactive oxygen species can, of course, be reduced by decreasing the 0 2 concentration available to interact with redox agents and excited states produced by photochemistry. This is a let-out which is clearly not available to organisms exposed to hyperoxia. The first line of defence to hyperoxia is to minimize the rate of production of reactive oxygen species. The extent to which this occurs at either the acclimatory (phenotypic) or adaptive (genetic) levels is not clear; evidence on the acclimatory or adaptive changes of redox interactions with 0 2 leading to reactive oxygen species as a function of hyperoxia is not, apparently, available (Halliwell and Gutteridge, 1989). Similarly, not many data are available on acclimatory or adaptive changes in the interaction of excited, triplet states of chlorophyll with 0 2. Here the quenching of the triplet states by carotenoids (e.g. /3-carotene) can preempt the formation of singlet 02, while/3-carotene per reaction centre, or per total pigments, increases with photon flux density for growth, the information on response to 0 2 is less clear (Siefermann-Harms, 1987). We might expect the/3-carotene content of cells of C 4 plants would be higher in those cells which accumulate 0 2 than in those which do not; however, conclusive data on this point seem to be lacking. Once the reactive oxygen species have been formed, they can be at least partly destroyed before they can do damage by the occurrence of

J.A. RAVEN

various enzymes, and of scavenging and quenching agents (see Table 2). H 2 0 2 can be destroyed by catalase, which converts it to H 2 0 plus 02, or by various peroxidases (ascorbate peroxidase in most O2-evolvers; glutathione peroxidase in some) which oxidise the reduced form of a co-substrate of the enzyme (Price and Harrison, 1988; Halliwell and Gutteridge, 1989; Stabenau et al., 1989;). O~ can be destroyed by dismutation to H 2 0 2 and 0 2 using one of the various kinds of superoxide dismutase (Halliwell and Gutteridge, 1989). The "OH formed from 0 2 or H 2 0 2 which have not been completely removed is extremely toxic; it can, however, be detoxified by anti-oxidants (scavengers) such as a-tocopherol (Hailiwell and Gutteridge, 1989). Finally, any singlet oxygen which is formed can be (in part, at least) quenched by/3-carotene; thus, /3-carotene has two chances to decrease singlet 0 2 toxicity, one in reducing its formation and the other in destroying it after its formation (Halliwell and Gutteridge, 1989). Data relevant to the role of those enzymes, quenchers and scavengers in increasing tolerance to hyperoxia in plants is scarce. However, there is an increased catalase activity in an 0 2 - tolerant mutant of a terrestrial C 3 plant (&Tcotiana tabacum) (Zelitch, 1989, 1990). The phenotype of the mutant is consistent with a reduced rate of CO 2 production in photorespiration due to a reduced H 2 0 2 dependent decarboxylation of glyoxylate and hydroxypyruvate, i.e. the mutant lowers 0 2 inhibition of photosynthesis bringing it closer to the 'theoretical' expectations for a C 3 plant (Edwards and Walker, 1983). The Nicotiana data do not bear immediately on more general tolerance to high 02, i.e. tolerance unrelated to consequences of RUBISCO activity. Catalase is, however, not a prerequisite for 0 2 tolerance, since it is absent from members of the Prasinophyceae (Micromonadophyceae) (Stabenau et al., 1989); at least some of these organisms have a CO 2 concentrating mechanism (Raven and Johnston, 1991) and so presumably accumulate O 2 to higher levels than are found in the surroundings. Presumably peroxidase(s) function in H 2 0 2 removal in these cells, although they lack glycolate dehydrogenase which does not produce H 2 0 2 in

P L A N T R E S P O N S E S T O H I G H O~ C O N C E N T R A T I O N S

many phototrophs. Instead, any glycolate which is produced is these organisms despite the CO 2 concentrating mechanism is oxidized by glycolate dehydrogenase which does not produce H 2 0 2 (Stabenau et al., 1989). Furthermore, Foster and Hess (1980) show that 48 h exposure of Goss,vpium hirsutum L. leaves to 75 kPa 0 2 in the presence of air CO 2 levels does not involve an increase in catalase activity, although glutathione reductase, and peroxidase, two components of an alternative pathway for H 2 0 2 disposal, do show increased activity. A mutant of Chlorella sorokiniana Shih. and Krauss which is tolerant of high O 2 has 3.5 times the superoxide dismutase activity of the wild type (Pulich, 1974). The differences in growth response of the wild type and the mutant with various energy and carbon sources (CO 2 in the light, and glucose or acetate in the dark) show that the 0 2 tolerance of the mutant and the 0 2 intolerance of the wild type are not related solely to photosynthetic growth, although differences between glucose and acetate as organic C sources in the dark are as yet unexplained (Richardson et al., 1969; Pulich and Ward, 1973). Furthermore, photosynthetic cultures were supplied with 2 kPa CO 2, so the involvement of a CO 2 pump, and of 0 2 accumulation within cells, is unlikely. Pirt and Pirt (1980), using Chlorella L,ulgaris Beijerinck, showed that gradual increase in 0 2 concentration (increments of less than 10 kPa) permitted growth of photosynthetic cultures with the same specific rate of growth, growth energy efficiency, and maintenance costs with 80 kPa 0 2 as with 20 kPa O 2. Whether these responses reflect a slow acclimation, or a selection of genotypes in the cultures, is not clear. A parallel between the 0 2 tension to which organisms are exposed and their superoxide dismutase activity has also been shown in a phototrophic context in studies on Symbiodinium-invertebrate symbioses (Dykens and Shick, 1982; Dykens, 1984; Shick and Dykens, 1985). Here superoxide dismutase activity is higher in photosynthetically more active symbioses, and, where it has been tested, superoxide dismutase activity parallels higher tissue O z concentrations in illuminated organisms. Supplying high 0 2 concentra-

27

tions to organisms in the dark also increases the superoxide dismutase activity. The data available for phototrophs shows that exposure to high 0 2 for varying times, with or without documentation of acclimation or adaptation to high 0 2, is related to an increase in one or more of the enzymes involved in H 2 0 : or O7; removal. However, data on transgenic Drosophila (fruit-fly) and on cultured flowering plant cells (Lycopersicon. Nicotiana) show that increased Cu-Zn superoxide dismutase activity does not invariably yield an increased 0 2 tolerance (Seto et al., 1990; Tepperman and Dunsmuir, 1990. These data may be explained, at least in part, by the finding (Yim et al., 1990) that isolated Cu-Zn superoxide dismutase generates 'OH during its slow inhibition by H 2 0 > This effect was not observed for the mitochondrial Mn superoxidc dismutase (Yim et al., 19911). The final defence against toxic effects of hyperoxia is repair of damage caused by the reactive oxygen species. This is not a very well investigated area. However, if the destruction of the D~ polypeptide of photoreaction two in classic 'photoinhibition' is indeed a function of reactive oxygen radicals (Kyle, 1987; Asada and Takahashi, 1987), then the well-investigated removal of damaged D I protein and its replacement with newly synthesized protein (Adir et al., 1990; Ohad et al., 1990) can serve as an example of repair of damage caused by reactive oxygen species (Raven and Samuelsson, 1986; Raven, 1989). To summarise the findings on the effects of reactive ox'ygen species in relation to hyperoxia, there is evidence for enhanced acclimatory and adaptive ability to detoxify reactive oxygen species as a result of short- and long-term exposure to high O 2. Data on the possibilities of decreasing the rate of synthesis of reactive oxygen species under hyperoxia from the expected value based on the rate under normoxia, and of an increased capacity for damage repair under hyperoxia relative to normoxia, appear to be lacking. However, what few data are available suggest that the acquisition of enhanced 0 2 tolerance need not incur either a decrement in growth rate, or a decrement in the energetic efficiency of growth or in the energy costs of maintenance, relative to popu-

28

lations which are neither acclimated nor adapted to hyperoxia. This conclusion is drawn from data on Chlorella species (Richardson et al., 1969; Pulich and Ward, 1973; Pulich, 1974; Pirt and Pirt, 1980). Accordingly, it seems that, when 0 2 effects cia RUBISCO and nitrogenase are eliminated by growth in saturating CO 2 and on combined N respectively, tolerance of high O- need not incur measurable penalties in terms of growth rate under resource-saturated or under energy'limited conditions, or in terms of maintenance energy costs in non-growing conditions. However, it must be emphasized that energy-saturated and energy-limited growth rates of C 3 plants at air levels of CO 2 are diminished by hyperoxia via the effects of RUB1SCO (see above). Furthermore, the possibility that a growth rate penalty is incurred due to hyperoxia under conditions in which elements specifically involved in detoxifying reactive oxygen species are limiting growth, e.g. Cu, Zn, Fe and Mn are involved in various superoxide dismutascs, Fe in catalase, and Se in glutathione peroxidase, should not be discounted (Price and Harrison, 1988; Raven, 1990). Finally, the available data do not permit evaluation of the possibility of enhanced mutation rates due to unrepaired damage to DNA as a result of hyperoxia. Relevance of data on extant plants to previous high 0 2 episodes

Phmts with diffusit,e CO 2 entry followed by fixation catalysed by RUBISCO The data on extant phototrophs discussed above show that many photosynthetic organisms grow with at least twice the present air-equilibrium CO 2 concentration in their photosynthetic ceils during steady-state photosynthesis. The common thread linking these organisms (Table 1) is the presence in the cells of higher than air-equilibrium CO 2 concentrations, and (in some cases) the restricted diffusion of 0 2 to a bulk medium at air-equilibrium, with a high ( ~ 1 mol m 3) external concentration of usable inorganic C. These O, concentrations of at least twice those in an air-equilibrium 0 2 solution.

J.A, RAVEN

Translating this argument to a time when O 2evolving photosynthesis had evolved but the bulk external O2 concentration was close to zero as a result of consumption in Fe 2+ and S 2-. oxidation, there are data consistent with substantial atmospheric CO 2 partial pressures at that time. The argument based on the weak young sun and the presence of liquid water (as opposed to ice alone), and with CO 2 as the main greenhouse gas, suggest rather higher CO 2 partial pressures than does the argument based on weathering rates (Walker, 1985; Kasting, 1987). At all events, a high CO: concentration in air-equilibrium solution available to O2-evolving phototrophs is indicated (Rothschild and Mancinelli, 1990). Applying the arguments of Samish (1975), we have approximately inverse gradients of CO 2 and of O 2. These, even in an acid ocean as envisioned by Walker (1985), and an atmospheric partial pressure of CO 2 100 times the extant value of 35 Pa, i.e. a partial pressure of 3.5 kPa CO:, would give a dissolved air-equilibrium concentration at 25°C of 1190 mmol m 3 CO2. Even with RUBISCO having a low CO 2 affinity and a low CO 2:O 2 selectively factor, an intracellular steady-state CO 2 concentration of 500 mmol m -3 would be amply sufficient to repress o~genasc activity of RUBISCO, granted the O 2 concentration in the cells in the steady state of about (1190-500) or 690 mmol m -3 suggested by the Samish (1975) arguments if the external O, concentration is zero. Such a situation, requiring a substantial diffusion limitation of CO, entry, would nevertheless be consistent with the natural abundance 13C/12C ratio of organic C at that time (Schidlowski, 1987; Raven and Sprent, 1989). We note that, based on studies on extant algal mats, 1 mm of sediment could give a steady-state 0 2 concentration in the sediment which is 500 mmol m -3 or more in excess of that in the bulk medium (Revsbech et al., 1981), albeit with H C O ; at seawater concentrations as the inorganic C source permitting this accumulation. The 1 mm depth of diffusion-restricting sediment necessary to give this 0 2 build-up would have been very helpful in restricting UV penetration to the cells in the absence of an 0 3 screen; suitable components of the screening/diffusion-limiting layer would be Fe corn-

PI AN'I' RESPONSES TO HIGH O: CONCENTRATIONS

pounds, e.g. Fe 3+ oxides precipitated by the evolved O, interacting with dissolved Fe 2(Raven and Sprent, 1989). These considerations suggest that benthic (but not planktonic) representatives of the early 0 2evolvers could have had substantial O, accumulation in illuminated cells, with clear implications for the generation of toxic O radicals. It is thus possible that at least some early OE-eVolving cells could have suffered from a high potential UV flux in the absence of a UV screen requiring atmospheric 0 2 to generate 0 3, while also being subject to intracellular 0 2 accumulation. We note that in the (eukaryotic) micro-alga Chlorella sorokiniana, an oxygen-resistant strain with enhanced superoxide dismutase activity is also more UV-resistant than is the wild type (Pulich, 1974). In addition to the need for mechanisms for detoxication of toxic O species, it is possible that OE-dependent biosynthetic pathways could have become established quite early in the evolution of OE-eVolving plants. Some of these biosyntheses lead to UV -screening organic compounds (Chapman, 1985). With the general trend toward lower atmospheric CO, partial pressure and lower equilibrium solution concentration (Berner, 1990) the possibility of 0 2 accumulation within aquatic plants declined. However, the absolute intracellular concentration in illuminated cells need not have declined, since the finite (but temporally variable: Berner and Canfield, 1989) external 0 2 concentrations which developed would have to some degree offset any decrease in the excess of intracellular over extracellular 0 2. Furthermore, the occurrence of finite external 0 2 concentrations permits the development of continuous (as opposed to daylight only) respiration. This trend of decreasing CO 2 and increasing 0 2 was probably a major selective pressure favouring the evolution of C 4 photosynthesis (Smith, 1976) and, by analogy, the various inorganic carbon concentrating mechanisms found in aquatic phototrophs and possibly, the CAM syndrome (Raven and Sprent, 1989): see below. However, it is important to remember that the great majority of extant terrestrial plants, and an even greater fraction of those in the past, have C 3

29

metabolism, i.e. depend on diffusive CO: entry to the active site of RUBISCO as well as on diffusive 0 2 loss from thylakoids to the bulk medium (see above). This mode of photosynthesis apparently characterized the earliest vascular land plants (Raven, 1977b,1984b,1985; Raven and Sprent, 1989). Extant C 3 plants can clearly compete well in the present atmosphere, as they could in the pre-industrial interglacial atmosphere with ~ 2 8 Pa CO, and 21 kPa 0 2, and even the glacial atmosphere with ~-21) Pa CO~ and 21 kPa 0 2. ls it likely that the terrestrial plants would have encountered lower atmospheric molar CO 2 : 0 2 ratios than the glacial value of ~ 10 37 Taking lhe highest 0 2 estimate of Berner and Canfield (1989) which relates to the late Carboniferous, a CO 2 : 0 2 of 10 -~ would need a CO z partial pressure of 35 Pa, i.e. the same as today. Thus, unless the CO 2 partial pressure in the late Carboniferous were lower than that found today, the C 3 plants would not have been exposed to a CO 2 : O~ ratio lower than that in the last glaciation. Berner (1990) suggests that late Carboniferous CO, partial pressure was about the same as the present value. While absolute values of gas concentrations are clearly important, especially for 0 2 toxicity considerations in the case of higher 0 2 partial pressures, we conclude that C~ plants are unlikely to have been seriously disadvantaged with respect to photosynthesis and its interactions with resource availability in the past relative to their extant, or even their last ice age, situation. While discussing C 3 terrestrial plants, it is important to examine the validity of implicit or explicit assertions that Or is more toxic to terrestrial (from the context, (i 3) than aquatic plants because of direct exposure to the atmosphere rather than to dissolved 0 2 (e.g.p. 38 of Chapman, 1985). The principal toxicities which this paper addresses relate to the intracellulur generation of toxic O species, involving reactions whose rates increase with increasing O~ concentration. Following the arguments of Samish (1975) and Raven (1977a,b,1984a,b,1985), we would expect a lower intracellular 0 2 concentration in non-photosynthesising tissues of aquatic than of terrestrial plants, but a higher intracellular 0 2 concen-

31)

tration in photosynthesizing tissues of aquatic than of terrestrial plants, granted diffusive CO 2 and O~ fluxes in all cases, and a medium of the extant atmosphere for terrestrial plants and of air-equilibrated solution for the aquatics. The argument essentially rests on the greater diffusion limitations in water than in air. Even if we consider organisms with CO 2 concentrating mechanisms (C 4 or CAM biochemistry and anatomy, or active CO 2 or HCO 3 transport), Table 1 shows that the achieved 0 2 accumulation in the steady-state of photosynthesis is generally at least as high in aquatics as in terrestrial phototrophs. It is clear that the generalisation of a greater 0 2 build-up in terrestrial plants, with a correspondingly greater rate of generation of toxic O species, is not tenable. However, there remains the possibility that the generation of toxic O-containing species in an atmosphere with a given 0 2 concentration has a greater effect on terrestrial plants than the effect of the generation of reactive oxygen species in a waterbody at equilibrium with the given atmosphere on aquatic plants. This possibility is very difficult to evaluate, especially in a geological context in view of the recent anthropogenic changes which make extant processes a less useful model for past events. Thus, a comparison of atmospheric toxicity via tropospheric 0 3, NO x and SO 2 to early land plants with aquatic toxicity via H~O~ and oxidized organic radicals to contemporaneous aquatic plants is not currently possible.

Evolution of CO2 concentrating mechanisms: timing and selection pressures Here we discuss the evolution, presumably in relation to a low absolute C O 2 concentration, a high O 2 / C O 2 concentration ratio and/or restricted availability of N, Fe, Mn or (on land) water, of 'CO 2 concentrating mechanisms' in the form of C 4, CAM, or active transmembrane entry of inorganic C (Smith, 1976; Raven et al., 1985; Griffiths, 1988; Raven, 1990). While it is very likely that the absolute 0 2 concentration in the environment, or even the 0 2 : CO 2 concentration ratio, are not the sole or even, necessarily, the

J.A RAVEN

main selective pressures leading to 'CO 2 concentrating mechanisms', the timing of the evolution of the various mechanisms would be of interest in terms of long-term variations in the atmospheric 0 2 concentration, and in the O 2 : CO 2 concentration ratio (Berner and Canfield, 1989; Berner, 1990). This interest is increased by the occurrence of substantial O2 accumulation in the tissues of organisms with CO 2 concentrating mechanism; the total 0 2 in the tissue is a function of external 0 2 concentration and the accumulation (concentration inside minus that outside) of 0 2 in the tissues. The earliest direct evidence for C 4 metabolism comes from anatomical and 13C/12C ratio data o n a n extinct panicoid grass (Nambudiri et al., 1978), now known to be of late Miocene age (about 10 Ma) and named Tomlinsonia thomassonii Tidwell (Tidwell and Nambudiri, 1989). An earlier origin of C 4 metabolism, which is polyphyletic even within single flowering plant families, can be inferred from the present distribution of C 4 members of a family and the palaeogeographical record (Osmond, et al., 1980). The direct morphological-anatomical and ~3C/12C ratio data for the past occurrence of CAM is even less ancient than is that for C4, i.e. 10,000 yr, for Opuntia polycantha Haw. (a facultatire CAM plant, i.e. able to carry, out C~ metabolism when supplied with ample water: Troughton et al., 1974). Indeed, ~ C / J 2 C data on Opuntia polvcantha dated at older than 40,000 yr shows that it was not carrying out CAM, although independent evidence is consistent with this being a result of a switch to C3 metabolism in a wetter environment. As with C~ plants, phylogenetic and palaeogeographical evidence suggests greater antiquity for this very polyphyletic pathway, possibly Palaeogene (like the C 4 pathway) in angiosperms. It would be of interest to determine when CAM evolved in the lycopsids and the pteropsids (Griffiths, 1988; Raven et al., 1988), both of which are substantially older than the flowering plants. This is particularly interesting for the lycopsids; CAM is widespread in submersed species of Isoetes, and the Isoetes clade of the lycopsids can be traced back to the upper Carboniferous, albeit in the form of terrestrial

PLANT RESPONSES TO H I G H 0 2 CONCENTRATIONS

plants (Raven et al., 1988). Interpretation of CAM in relation to atmospheric 0 2 : CO 2 concentration ranges in lsoetes is complicated by the fact that even amphibious/terrestrial members such as the astomatous Isoetes (formerly Stylites) andicola Amstutz., Isoetes andina Hook and Isoetes nocogranadensis Fuchs, as well as all submersed species tested, obtain their CO 2 via their roots from the CO2-rich sediments (Raven et al, 1988). Even fewer anatomical and ~3C/~2C ratio data are available to indicate the past occurrence of the diverse, polyphyletic CO 2 concentrating mechanisms found in many aquatic plants and some terrestrial algae and lichens (see above; Raven et al., 1990). Raven (1987a) and Raven and Sprent (1989) suggest that the increased 13C/12C in marine sediments from the end of the Cretaceous onwards is consistent with an increased representation of phototrophs with 'CO 2 concentrating mechanisms'. However, other explanations are equally possible (Raven and Johnston, 1991). Such evidence as is available, then, is consistent with the occurrence of the C 4, CAM and aquatic inorganic C pump modes of CO 2 accumulation since at least the Palaeogene. Between the Palaeogene and today the O 2 mass in the atmosphere has generally been above the extant value (fig. 13 of Berner and Canfield, 1989). The analysis of Berner (1990) suggests a steady fall in atmospheric CO 2 mass during the Tertiary from a late Cretaceous value of twice the present CO 2 mass to a value close to that found today. This, with the 0 2 data, is consistent with increasing selective pressures favouring CO 2 concentrating mechanisms since the end of the Mesozoic.

02 concentrations, mutation rate and phenotypic damage accumulation Here we address the relationship between 0 2 concentration and mutation rate (Imlay and Linn, 1988) and of damage accumulation in the phenotype, e.g. the destruction of long-lived proteins which are difficult to replace in vivo (Dunn et al., 1989). The effects are, of course, special cases of 0 2 toxicity, but are given separate treatment here because of their perceived importance in deter-

31

mining genetic variability in offspring in the case of DNA modification, and in damaging irreplaceable and not readily repaired plant components, such as the enucleate sieve elements in the phloem of long-lived monocotyledons which lack secondary thickening (Raven, 1991). Such considerations also bear on the role of 0 2 toxicity in aging (Bell, 1988; Lovelock, 1989). In considering damage to DNA by reactive oxygen species it is convenient to distinguish organisms in which all cells can divide (unicellular organisms, colonies) from those in which new organs, and at least sexually produced progeny, are derived from a small subset of cells in dividing, growing parts of the plant. In the unicells and colonies, disadvantageous genotypes are eliminated by (infrequent) sex (Bell, 1988), and by competition, while in multicellular organisms there is often an editing mechanism in the growing regions which rejects disadvantageous genotypes (Klekowski and Kazarinova-Fukshansky, 1984 a,b; Klekowski, 1988). Unicellular organisms, especially if they are non-mobile, cannot do a great deal about the 0 2 concentration to which their DNA is exposed. In the light, a photosynthetic unicell has an intracelIular 0 2 concentration which exceeds that in the medium to an extent which varies with inter alia the occurrence of a CO 2 concentrating mechanism (Table 1). In natural light-dark cycles there is more or less evidence of phasing of the cell cycle with cell division occurring in the dark; the extent to which this minimizes 0 2 access to DNA at particularly sensitive times of the cell cycle deserves further investigation. At all events, sex seems to be an important way in which the genetic integrity of unicells is maintained (Bell, 1988). Might the frequency of sex in, for example, the diatoms be a function of the mean intrace[[ular 0 2 concentration? More work is needed. In multicellular organisms, where individuals are generally larger, longer-lived and less numerous then for unicellular organisms, there are a number of characteristics which recur in independent evolutionary approaches to multicellularity, and which may contribute to reducing 0 2 damage to the DNA which contributes to the progeny. One of these is the tendency to localize

32

cell division into growing points/meristems, and a subsidiary characteristic is the tendency to have few, or no photosynthetically competent plastids in these growing regions even when they are in the light. The literature which documents localization of growth is well summarised in reviews and monographs (Bold and Wynne, 1985, for algae; Mischler and Churchill, 1985, and Crandall-Stotler, 1986 for bryophytes; Gifford and Foster, 1989, for vascular plants). These monographs and reviews make less of the tendency to reduce or eliminate photosynthetic capacity from growing regions, but references which they cite clearly show that reduction or elimination occurs in many clades of multicellular plants, and is even a feature of highly differentiated acellular macroalgae such as Caulerpa. Localization of cell division and of extension (elongation) growth seem to be prerequisites for the production of a highly differentiated organism. Reduction in, or elimination of, photosynthetic capacity in growing regions may be counter-productive energetically, in that computations of the energetics of ATP and NADPH for plant growth based on carbohydrate as the C source show that less C may be lost as CO 2 if illuminated plastids generate the ATP and reducrant (Raven, 1976). The relative absence of CO 2 fixation in growing regions of terrestrial plants means that their water loss in transpiration is low. A minimal water loss from non-photosynthetic organs is essential if the whole plant is to maximize dry matter increment per unit of water lost, but it does lead to problems of distribution of some soil-derived nutrients which are mobile in the xylem but not in the phloem, such as B and Ca. The xylem delivers these elements to the sites of transpiration, i.e. where most photosynthesis occurs, in mature non-growing organs, while the net requirement for them is in weakly transpiring growing regions (Raven, 1977c,1980,1984b,1986). The separation of growth from photosynthesis poses what, teleologically, appear to be a problem for the plant, with various anatomical and physiological solutions (Raven, 1977c,1980,1984b,1986) What compensatory advantages might the organism obtain by separating the main sites of growth from those of photosynthesis? Appeals to

JA. RAVEN

the off-invoked advantages of differentiation and division of labour should, when possible, be made quantitative. This is possible for cell division rate: empirical evidence, in agreement with theoretical considerations, shows that unicellular eukaryotes growing photosynthetically have minimum generation times which are twice as long as those of non-photosynthetic organisms of a similar size (Raven, 1987b). Accordingly a given volume of macrophyte growing region could grow twice as fast if it were non-photosynthetic as a result solely of the solution of catalysts of heterotrophic processes by catalysts of photosynthesis in a hypothetical photosynthetic growing region. Furthermore, constraints imposed by the need to supply light, external CO 2, and liquid water to photosynthesizing cells would exacerbate the growth rate effect and complicate the morphogenetic processes. In addition to these rationalisations of the tendency to minimize photosynthesis in growing zones of macrophytes we can add the possibility of maintaining a low 0 2 concentration in these growing zones, thus reducing 0 2 damage to the DNA which will be most directly involved in producing progeny. Land plant sporophytes have gas spaces ramifying through the tissues with the exception of vascular tissue and the parts of the growing regions where cell division occur (Canny, 1973; Roland, 1978). The combination of high metabolic rates in growing regions, the absence of photosynthetic O 2 production in these regions, and their minimal provision with gas spaces, mean that O 2 supply rate equal to the O 2 consumption rate is achieved with a significantly lower than air-equilibrium steady-state O. concentration in the growing regions, especially in the bulkier apical regions. This conclusion implicitly requires that the 0 2 partial pressure in the intercellular gas spaces closest to the growing regions contains O 2 at close to, or below, the atmospheric partial pressure. This is plausible for C 3 plants, and for C 4 plants of the PEPck and NADme subtypes, where the high 0 2 in illuminated RUBISCO-containing cells is only transmitted to the rest of the plant via solution in xylem and phloem adjacent to these O2-evolving cells. The 0 2 lost to the gas

PLANT RESPONSES TO ttlGH O, CONCENTRATIONS

spaces is lost through the stomata which are open during net CO 2 uptake; no appreciable gaseous 0 2 accumulation occurs. Even with 550 mmol m 3 02 in phloem sap leaving illuminating photosynthetic tissues of C 4 plants of the PEPck and NADme subtypes (Table 1), and retention of all of this 0 2 en route to a growing region (cf. Raven, 1977c), the ratio of 0 2 to organic C in the phloem sap is on the order of 0.55:6000 (Raven, 1977c). With one-third of the organic C supplied to a growing tissue being consumed in respiration (Raven, 1976), the 0 2 transported in the phloem only supplies 0.55 mol m -3 of the 6000/3 or 2000 mol m -3 0 2 needed to assimilate the organic C in phloem sap, i.e. only 2.75.10 -4 of the requirement. This is negligible. More difficult to rationalise in terms of minimizing 0 2 build up in growing regions are plants performing CAM, and in many vascular (and other) aquatics. Here there is significant 0 2 build-up in internal gas spaces in the light behind the closed stomata of plants performing CAM, and in gas spaces of aquatics (Table 1). 0 2 partial pressures of 40-60 kPa are not uncommon here, while prolonged darkness gives partial pressures below 20 kPa. It is not easy to see how growing regions could receive adequate 0 2 by aqueous phase diffusion from the terminii of the gas spaces to support respiration and growth in the dark without being subjected to steady-state 0 2 concentrations in the cells which are very substantially above the air-equilibrium level when the plant is illuminated. The alternative of an 0 2 supply to growing regions in light which maintain a steady-state 0 2 concentration below the airequilibrium value, and anoxia in these regions in the dark, with a corresponding lack of growth, has apparently not been examined for CAM plants. However, the occurrence of root growth when the shoot is illuminated and an absence of growth associated with root anoxia when the shoot is darkened is the normal in situ situation for the (non-CAM: Raven et al., 1985) seagrass Zostera marina (Pregnall et al., 1984; Smith et al., 1984). This mode of growth is presumably associated with post-anoxic toxicity in the roots on a daily basis (see Monk et al., 1989). Distinguishing fermentation by growing points when the plant over-

33

all is aerobic and producing organic acids, from dark acidification in CAM, is not as easy as it may seem (Keeley, 1988). Similarly, the possibility that an aqueous diffusion barrier could change its resistance (as happens in legume root nodules: Dakora and Atkins, 1990b), so as to restrict 0 2 access to growing regions in the light, has not been examined. Thus, there are possibilities whereby 0 2 could be prevented from accumulating in meristems to higher than air-equilibrium values in the light in CAM, and in certain aquatic plants, in air or in air-equilibrium medium, although they need testing. A final case which is much more difficult to rationalise in terms of 0 2 concentrations in growing regions which is invariably below the air-equilibrium value is that of aquatic plants in rock pools, sediments and very productive freshwaters where the external 0 2 concentration may reach several times air-equilibrium (Table 1). Maintaining growing region 0 2 at subambient levels in light or dark is difficult here; the finding that dark respiratory. 0 2 uptake in some aquatic macrophytes is not saturated by air-equilibrium 0 2 in solution (Chapter 6 of Raven, 1984a) does, however, lend credibility to some (unspecified) barrier to 0 2 diffusion which might serve to restrict 0 2 supply to growing regions. However, this is in need of much more investigation. An interesting observation by Pefiuelas (1987) may be significant here: growth of the submerged freshwater angiosperm Potamogeton crispus L. under hyperoxic conditions decreases the fraction of the plant occupied by gas spaces, a circumstance which could regulate the 0 2 partial pressures in meristems. Our conclusion o n O 2 concentrations in growing regions of extant plants with CO 2 concentrating mechanisms is that in some cases (C 4) the growing regions can maintain 0 2 concentrations below air-equilibrium values, while in terrestrial CAM, and many aquatic plants such maintenance of low O 2 concentrations in growing regions is not impossible but has not been demonstrated. What of plants growing at times when 0 2 was higher than the present atmospheric partial pressure? There is little theoretical difficulty in conceiving of barriers to 0 2 diffusion in cell walls of

34

growing regions without impeding cell expansion and cell division; these could maintain 02 concentrations in growing cells which are similar to the extant values, provided that the O 2 is supplied to the growing regions at a constant concentration. Variations in 0 2 supply on a diel basis (e.g. in CAM) create problems similar to those discussed for extent CAM plants, t Our discussion shows that maintaining low 0 2 concentrations within growing regions of differentiated plants is not theoretically impossible, even with external O E partial pressures higher than the ambient values (cf. Berner and Canfield, 1989). However, it is by no means clear that such low O 2 concentrations are, and were, maintained. If they were not so maintained in growing regions, enhanced mutation rates could be expected. However, many plants have growing regions with an editing mechanism which restricts continued division of deleterious genotypes and their perpetuation in the cells contributing most directly to progeny (Klekowski and KazavinovaFukchansky, 1984a,b; Klekowski, 1988). However, we know nothing of the effect of external 0 2 partial pressure on mutation rates of plants; only recently have data been presented on mutation rate per year in annual as opposed to perennial plants in the extant atmosphere (Klekowski and Godfrey, 1989). Less immediately significant in restricting mutation rates on a generation to generation basis, but very important for maintaining fitness of the phenotype of an individual, is the effect of high intracellular O E partial pressures on the functioning of vegetative cells. Such cells include RUBISCO-containing cells of C 4 plants, and the majority of vegetative, or at least photosynthetic, cells of terrestrial CAM plants and many aquatic macrophytes. Longevity of the RUBISCO-containing cells of C 4 plants is generally only a few months, so damage can be tolerated in the short term before the photosynthetic organ is discarded. The cells exposed to high O 2 in the other categories of plants listed above are both a larger fraction of the total number of cells in the plant, and at least some of the cells are much longerlived. Tolerance of damage which can restrict resource acquisition, retention and use in enhanc-

J,A, RAVEN

ing fitness a n d / o r damage repair, at the expense of resource diversion, must take place. Higher 0 2 partial pressures in the past would have exacerbated these effects as well as increasing the 0 2 toxicity to vegetative cells of C 3 terrestrial plants, especially in photosynthetic tissues. Raven (1991) suggests that long-lived enucleate phloem conducting cells in perennial monocotytedons lacking secondary thickening, are particularly at risk from OE-related damage since the cells cannot be replaced, and lacking nuclei, may be difficult to repair. However, it is likely that these organisms evolved in the Cretaceous or possibly the Jurassic, which is well after the highest Phanerozoic 0 2 partial pressures suggested by Berner and Canfield (1989).

Conclusions Many extant plants can tolerate 0 2 concentrations in at least some of their cells for some of their life cycle which are higher than the highest 0 2 concentrations found in C 3 terrestrial plants in the present atmosphere. Some of the earliest OE-eVolving phototrophs in the high CO 2 conditions to which they were exposed could have built up 0 2 in microbialites or microbial mats to greater than present air-equilibrium values even before O E accumulated to a significant extent in the atmosphere. Thus, many OE-eVolving phototrophs can now, and have for most of their existence, tolerated 0 2 concentrations substantially in excess of the present air-equilibrium value related to O 2 build-up in their cells and tissues during photosynthesis. This suggests that the upper limits on Phanerozoic 0 2 content of the atmosphere (Berner and Canfield, 1989) would not have been a major constraint on the terrestrial biota at least as far as plants are concerned (cf. Lovelock, 1989). However, the costs to growth rate, productivity, reproductive potential and the capacity to survive adverse environmental conditions of tolerating, or repairing, damage inflicted by high environmental O 2 has not been quantified. Fire could well be a more important constraint on the nature of terrestrial biota as a function of increased atmospheric 0 2 content than are the

PLANT RESPONSES TO H I G H 0 2 CONCENTRATIONS

direct effects of 0 2 on the physiology of live plants (see Chaloner, 1989, and Robinson, 1989).

Acknowledgements Stimulation and encouragement by colleagues, especially Profs. W. G. Chaloner, F.R.S., and J.I. Sprent, and Drs. D. Edwards and R. Parsons, is gratefully acknowledged. Work in JAR's laboratory on inorganic C acquisition by algae is supported by NERC, and that on N 2 fixation, and on NH 3 assimilation, is supported by AFRC.

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