Shifts in carbon isotope ratios of two C3halophytes under natural and ...

Oecologia

Oecologia (Berl.) 44, 241547 (1980)

9 by Springer-Verlag 1980

Shifts in Carbon Isotope Ratios of Two C3 Halophytes Under Natural and Artificial Conditions Robert D. Guy 1,, David M. Reid 1, and H. Roy Krouse z Biology Department and 2physics Department, University of Calgary, Calgary, Alberta, T2N 1N4, Canada

Summary. The total carbon 313C values of two C s halophytes, Salicornia europaea L. ssp. rubra (Nels.) Breitung and Puccinellia nuttalliana (Schultes) Hitch., native to inland saline areas of Alberta, Canada, were determined for plants grown under controlled conditions of supplied NaC1 in the nutrient solution, and for plants found growing in the field. Field specimens were collected along line transects which ran from areas of high salinity to areas of low salinity across the pattern of species zonation. The 313C value of the two species seemed to reflect the water potential of the soil (if~oi~)as measured arbitrarily at a depth of 10 cm, becoming less negative as the if~w ~ decreased. Over a linear distance of 5.55 m, S. europaea ssp. rubra showed a shift of +5.3~ as the if soil went from - 2 5 • 102 kPa to a minimum of - 7 3 • 102 kPa. For P. nuttalliana, the 313C values differed by 3.4O/0o over a distance of 7.45 m where the maximum difference in if~w ~ was 12.7 x 102 kPa. However, 613C values of P. nuttalliana only roughly reflected the spatial trends in if~o~ at the time of collection. In the growth chamber, the 313C value of S. europaea ssp. rubra changed by a maximum of + 8.0~ when the solute potential of the nutrient solution (if~oln) was dropped from --0.25• 102 kPa to -64.25 • 102 kPa; while the cSa3C value of P. nuttalliana changed by a maximum of + 10.8~ when the if~oln was dropped from -0.25 • 102 kPa to -40.25 • 102 kPa. Linear regression analyses indicated that the 613C values of both species were strongly correlated (P < 0.1%) with if~an. The observed shifts in 612C may represent changes in the mode of photosynthetic CO2 fixation. However, a number of other explanations, some of which are discussed in the text, are also possible. A proper ecophysiological interpretation of such shifts in 613C values of C 3 plants awaits a better understanding of the isotope fractionation mechanisms involved.

Introduction Over the past decade, a considerable body of literature has appeared establishing the use of the stable isotope ratio, ~3C/12C, as a reliable means by which the photosynthetic mode of higher plants, C 3- or C,-pattern, can be determined. C3 plants have bl3C values (expressed in per mille units relative to the standard, Pee Dee Belemnite) of - 2 3 to -32o/oo, while C4 plants have values of - 9 to - 16~ (Bender, 1971 ; Smith and Epstein, 1971 ; Troughton, 1971). Fractionation occurs at the initial carboxylation sites of ribulose-l,5-biphosphate (RuBP) carboxylase in C 3 plants (Park and Epstein, 1960) and phosphoenolpyruvate (PEP) carb*

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oxylase in C, plants (Whelan et al., 1970; Whelan et al., 1973). The differences in b13C result from differences in discrimination towards 13C by these enzymes (Benedict, 1978; Smith, 1972). The total carbon 613C value of any one C3 or C A species does not usually vary by more than one or two per mille, even when sampled from widely separated geographic locations (Smith and Brown, 1973). Crassulacean acid metabolism (CAM) plants on the otherhand, display very variable 613C values covering the full range of - 1 1 to -33O/oo (Bender et al., 1973). The 6~3C of any given CAM species is often subject to broad fluctuations largely dependent upon conditions of the environment (Bender et al., 1973). Values obtained presumably reflect the relative contributions to total plant carbon as fixed by RuBP carboxylase and/or PEP carboxylase (Bender et al., 1973; Lerman et al., 1974; Osmond et al., 1973). The use of 613C values has thus proved to be a useful tool for the measure of CAM as affected by photoperiod (Lerman and Queiroz, 1974), leaf age (Lerman et al., 1974), thermoperiod, drought and salinity (Osmond et al., 1973). Application of the technique to field situations has produced mixed results. In some cases (Eickmeier and Bender, 1976; Rundel et al., 1979; Szarek and Throughton, 1976) no evidence for substantial photosynthetic flexibility of CAM plants was obtained. Osmond (1975) found that in Australia, the nonendemic Opuntia inermis had more negative b13C values in the more mesic southern part of its range, but showed very little variation elsewhere. Osmond et al. (1975) were able to relate changes in 313C of Sempervivum spp. to the relative availability of water. To date, similar applications have not generally been possible with C3 and C4 plants because of the apparent constancy of their 613C values and a poor understanding of the processes involved (Lerman, 1975). Controlled conditions of temperature (Smith et al., 1973; Troughton and Card, 1975) and available oxygen (Berry et al., 1972) have been found to have only minor effect. Here we present evidence for comparatively large and predictable salt-induced differences in 613C among two species of C3 halophytes, Salicornia europaea L. ssp. rubra (Nels.) Breitung and Puccinellia nuttalliana (Schultes) Hitchc. The former is a succulent annual member of the Chenopodiaceae, while the latter is a non-succulent perennial grass. Both are native to highly saline inland salt marshes of western Canada. Tiku (1976) reported that the use of nutrient solutions containing NaCI caused a significant decrease in light compensation point in S. europaea ssp. rubra. No such effect was noted in Distichlis stricta, a C4 halophytic grass. It was in view of these results that we chose to investigate, by the stable carbon isotope method, the possibility of a salt-induced shift in photosynthetic patterns of S. europaea ssp. rubra and P. nuttalliana, in the field and under controlled conditions.

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Methods

Field Data During the summer of 1978 the vegetation of several inland saline areas was studied along a number of line transects laid out perpendicular to the observed pattern of species zonation. Plant material for isotopic analysis was collected on two of these transects, to be referred to here as A2 (for Puccinellia nuttalliana, collected September 1) and D2 (for Salicornia europaea ssp. rubra, collected August 10). Transect A2 was located on the shore of a saline slough 2.5 km southwest of Beiseker, Alberta (51~ 113~ and was subject to flooding earlier in the season. Transect D2 sampled the vegetation of a saline seepage area 15 km southwest of Standard, Alberta (51~ 113~ and was not subject to flooding at any time of the year. Healthy shoots were washed in distilled water, damped dry, placed in sealed plastic bags and quickly frozen on dry ice. Soil water potential (O~oil) was determined psychometrically before and after the periods of collection with a Wescor HR-33T microvoltmeter in combination with several Wescor PT51-05 soil hygrometers planted to a depth of 10 cm. As many shoots as possible were collected within a 5 cm radius of the point of hygrometer placement. Consequently, the number of individuals per sample varied from 1-3 for P. nuttalliana, and from 5-25 for S. europaea ssp. rubra. Frozen plant material was freeze-dried, then ground (S. europaea ssp. rubra, non-woody succulent portions only) or chopped (P. nuttalliana, leaf tissue only). Later in the season, transects were surveyed for topography and per cent cover of species was estimated within standard Daubenmire Plots (20 cm x 50 cm) at 0.5 m intervals along the transects. At transect D2, soils were sampled from pits and/or soil cores removed with a sharpened acrylic tube (5 cm bore) at 15 positions along the entire length of the transect (18 m). The cores were taken back to the lab and subsampled at depths of 0.25, 1.0, 3.0, and 5.0 cm, and every 5 cm to a maximum depth of 70 cm, for [I/soil determinations (dew point method) in Wescor C-52 sample chambers. All measurements were made at room temperature (20.1-21.2 C). Soil moisture percentage in the 2-12 cm zone was calculated on an oven-dry weight basis.

Growth Chamber Studies All plants were grown simultaneously in a single growth chamber (Sherer CEL 37-14) from collected seed sown on No. 1 granite grit (Imasco). Salicornia seeds were lightly scarified with sandpaper to insure a more even germination. There were 11-12 Puccinellia and 5 7 Salicornia plants per tray, suspended over a 6 1 volume of solution. There were 11 treatments in all, 1 tray per treatment (except at the 0 NaC1 level where 2 trays were used), randomly arranged. Sodium chloride was added to half-strength Hoagland's solution (Hoagland and Arnon, 1950), with an osmotic potential (~) of 0.25x 10 2 kPa, to give total O~olnvalues of -0.25, -2.25, 4.25, -8.25, - 12.25, 16.25, -20.25, -24.25, -32.25, 40.25, and -64.25 x 102 kPa. Plants were stepped up to their final NaC1 treatments through -0.25, -2.25, -4.25, - 8.25, 16.25, and - 32.25 x 10 2 kPa, the nutrient solution being changed every fifth day after the - 2.25 x 10z kPa level had been reached. Water levels were kept constant by the addition of distilled water as regulated by humidifier floats. Aeration with compressed air was employed to keep the solutions well mixed. Plants were maintained for 1%22 days after the highest salt concentration had been reached. 242

Thus, material for isotopic analysis was harvested after a total of 61 64 days growth. For Salicornia, 5-8 branch tips ( ~ 1 cm long) per treatment were pooled together for determination of shoot Ow (data not presented here). Branch tips varied in length somewhat because they were severed only at the stem joints. These branch tips were dried in an oven at 80~ C and ground for isotope analysis. In addition, 15 other randomly selected branch tips per treatment were collected and treated similarly. For Puccinellia, whole shoots were placed in plastic bags, frozen on dry ice and freeze-dried. Three intact green leaves per plant were chopped and pooled together from within each treatment tray. At the -64.25 x 102 kPa level, Puccinellia plants were severely stunted and not enough material was available for analysis. Daily temperature regime was programmed according to a sine curve approximating conditions expected in the field during the height of the growing season. Maximum temperature during the light period was 25.5~ C while the minimum night temperature was 10~ C. Illuminationwas provided by two banks of eight Sylvania gro-lux lamps staggered 0.5 h apart (16 h total photoperiod; maximum light intensity at the "soil" surface of 295 gE m. 2s-1). At some point, one bank of lights failed to turn off at night. This was not discovered until the end of the experiment. Humidity was not controlled but normally reached about 76% R.H. at night, dropping to about 46% R.H. in the afternoon. This compares favourably with field observations. Growth of plants was good, being comparable to what is encountered in the field under optimal conditions of low density and adequate soil moisture. Soil water potential (~w~ is a combined measure of osmotic or solute potential (Os) and matric potential @m). In the growth chamber studies, ~b~~ is essentially equivalent to ~o~n because the Om of damp granite grit is close to 0 x 102 kPa. In the field situations, the Om of the typically clay soils was, under most circumstances, probably only a minor component of the O~o~t as the soil moisture percentage was generally quite high. However, at transect D2 on the day of tissue sampling (Fig. 1b), ~*~ may have been more important. Carbon-Isotope Analysis: For each determination, CO2 was collected from the total combustion of 10 mg of tissue (dry weight) in a quartz ampule. The combustion line was similar to that of Troughton and Card (1975) but, in the end zones of the furnace, silver powder and manganese dioxide were present to react with halogens, sulphur compounds, and nitrogen oxides. Analyses were carried out on a mass spectrometer built around a Micromass 602 analyser. Ion currents were digitized by an Analogic AN5800 Series Computer Data Conversion System and 613C values obtained using a Texas Instruments 980A Computer. All field data are presented according to transect position to indicate associated spatial trends. The background 613C value of ambient growth chamber air was not determined but, as all plants were grown together, relative values are correct. However, for this reason, 6'3C values of growth chamber material can not be directly compared to those of field specimens, except in terms of magnitude and direction of change.

Results

The vegetation of saline areas of western Canada has been described by Keith (1958), Dodd and Coupland (1966), and Walker and Coupland (1970). Dodd and Coupland (1966) recognized seven halophytic plant communities, defined by their dominant species, occurring along a gradient from the depressed position to the dry edge of a saline area. Six dominants are important

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Fig. 1 A and B. Transect D2. A Per cent cover of Salicornia europaea ssp. rubra ( - - ) , Puccinellia nuttalliana ( - - - ) , and Hordeum jubatum (. . . . . ). B Change in tp~w ~ (10 cm) (o) and 313C of S.e. ssp. rubra (e) according to transect position. Soils were dry at the surface but moist at the depth of the hygrometers (August 10, 1978)

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Fig. 2A and B. Transect A2. A Per cent cover of Salicornia europaea ssp. rubra ( - - ) , Puccinellia nuttalliana ( - - - ) , Hordeum jubatum ( . - . - ) , and Triglochin maritima ( ..... ). B Change in ~oil (10 cm) (o) and 613C of P. nuttalliana (e) according to transect position. All soils were moist to the surface, the soil moisture percentage in the 2-12 cm zone being between 33 and 42% along the entire length of the transect on the day of collection (September 1, 1978)

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and are found in the following order: Salicornia europaea ssp. rubra, Triglochin maritima, Puccinellia nuttalliana, Distichlis stricta, Hordeum jubatum, and Agropyron spp. For the sake of reference, the per cent cover of species along the relevant portions of transects A2 and D2 is presented (Figs. 1 a and 2a). The tendency was for good development of S. europaea ssp. rubra on the most saline soils, with H. jubatum reaching almost 100% cover over much of the less saline areas. P. nuttalliana found its best development between these two dominants. The fi~3C values of Salicornia shoots closely paralleled the changes in ~,~7n at a depth of 10 cm (Fig. 1 b). As each determination was on material pooled together from several adjacent plants at each position along the transect, the observed difference of 5.30/oo over a mere distance of 5.55 m is quite remarkable. On transect A2 (Fig. 2b), ~ C values for Puccinellia did not show such a close agreement with the t)~~ (10 cm), but basic trends were the same. It is apparent that soil water potential at a single depth of 10 cm is probably not an adequate measure of the salinity to which a plant is exposed. This is particularly true as the depth

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Fig. 3, Transect D2 Topographic ~O~ ~ Profile. Isolines indicate areas of equivalent t/Sw(• 102 kPa). The position of dashed lines is somewhat conjectural. Elevation is relative to the soil surface at the 0 m position. Soil moisture percentage in the 2-12 cm zone was above 30% along the entire length of the transect, reaching a maximum of 54% at the 7.0 m position (October 19 20, 1978)

of rooting is not necessarily consistent from one individual to the next. Very marked differences in soil water potential are found at different depths of the soil profile and are subject to change during the growing season. A topographic kO~~ profile for transect D2 (Fig. 3) illustrates the kind of horizontal and vertical patterns possible under non-flooding conditions. At the time these soils were sampled the ground was damp to the surface, although a salt crust was present over the first 3 m of the transect. It is clear from this figure that overall soil salinity was highest at the 0 m position (D2-0.0), despite the more negative I//Sw~ (10 cm) for D2-1.0 observed at an earlier date when shoot material was collected (Fig. 1 b). This might explain the seemingly anomalous 613C value of -23.5~ at D2-0.0. Discrepancies between 0 ~ ii (10 cm) and ~$13C values of Puccinellia leaf material on transect A2 (Fig. 2b) might be similarly explained. For instance, the ~$13C value of - 2 6 . 4 % o at A~22.65 is less negative than might be expected, however, earlier in the season ~ i ~ (10 cm) at this position was relatively higher. Thus a "dilution effect" may be responsible, emphasizing the importance of plotting field data according to transect position. Although at any given point along 243

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To verify the field observation that the 313C values of the two C3 halophytes in question reflected the degree of salinization of the soil, plants were grown under controlled conditions where the only variable was the concentration of NaC1 in the growth medium. For both Salicornia (Fig. 4) and Puccinellia (Fig. 5) highly significant (P < 0.1%) correlations were obtained. Each data point in Fig. 4 is the average of two determinations on separate batches of pooled branch tips collected from the same set of plants. Within each treatment, these values differed by an average of 0.72~ The most negative 613C value obtained was - 34.9~ at -0.25 x 102 kPa, while the most positive value was -26.9O/oo at -64.25 x 102 kPa. Although the fitted least squares regression line is based on the data at all NaC1 concentrations, 6a3C values did not appear to be affected until a ~b~~ of at least - 16.25 x 10z kPa was reached. Maximum growth, on the otherhand, was obtained at - 8 . 2 5 x 102 kPa (data not presented). The magnitude of change of 6a3C values for Puccinellia was about 2.5 times greater than for Salicornia, being as much as 10,8~ over a O~an range of 40 x 10 2 kPa. In this case, 613C appeared to be affected at even the lowest level of NaC1 supplied (total ~,~a" of -2.25 x 102 kPa). Maximum fresh weight growth of Puccinellia occurred in the absence of NaC1 (data not presented). The larger effect that NaCI had on 613C of Puceinellia may reflect the lower degree of salt tolerance by this species, as compared to Salicornia. Despite the dramatic salt-induced shift in ~513C of Puccinellia under controlled conditions, the values obtained on transect A2 covered a range of only 3.4~ However, as the maximum difference in ~bS~ w (10 cm) was only 12.7 x 1 0 2 kPa, the magnitude of the observed shift is close to what would be expected, about 3.1%o as calculated from the slope of the fitted regression line in Fig. 5. Unfortunately, leaf material was not collected in the vicinity of A~18.0 where small amounts of Puccinellia were later found to be growing at a 0~oii (10 cm) of close to 0 x 102 kPa (Fig. 2a and b). At the time of collection, the Salicornia plants on transect D2 (Fig. 1b) were growing over a range of 0~w ~ (10 cm) of about 50 x l 0 2 kPa. From the slope of the fitted regression line in Fig. 4, we would expect a concomittant shift in 613C of about 4.8~ This compares favourably with the observed shift of 5.3~

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Fig. 5. Carbon isotope discrimination values of growth chamber grown Puccinellia nuttalliana according to ~bs of the nutrient solution. Values are not corrected for the isotopic composition of the growth chamber air a transect environmental conditions will vary tremendously in both time and space, basic relative patterns along its length will be more predictable. This is particularly relevant with regard to transect A2 where portions of the Puccinellia zone were flooded from mid-April until late June, 1978. 244

In the field, 613C values of whole shoot or leaf tissue of the two C3 halophytes, S. europaea ssp. rubra and P. nuttalliana, seemed to parallel spatial trends in 0~w ~ (Figs. 1b and 2b). Under controlled conditions in the growth chamber, the 613C values of these two species were highly correlated with the NaC1 concentration (expressed in terms of ~b~~176of the growth medium (Figs. 4 and 5). The amount of change was quite large, being as much as 10.8%o for Puccinellia. Differences might have been even more dramatic had determinations been based on the soluble tissue fraction rather than total plant carbon (cf. Lerman and Queiroz, 1974). To the best of our knowledge, differences in 613C within single species of vascular C3 plants along an environmental gradient under field conditions have not previously been reported. Osmond et al. (1975) could not detect any influence of water supply on 613C values of seven alpine species of Saxifraga. ShomerIlan et al. (1979) have, however, been able to correlate seasonal changes in 613C values of two species of lichen with monthly rainfall. Although variation in ~13C values of C3 plants is not likely to be as great as in plants possessing a flexible form of CAM, considerable potential for environmental studies does exist.

To this end it will be necessary to define, more precisely, the effects in the glycophytes Zea mays, Gromphrena globosa, radish, mechanisms of differential isotopic fractionation in C3 plants. and Triticum aestivum at NaCI concentrations of 0, t00, and In CAM plants, large positive shifts in the 613C discrimination 400 mM, and in the halophytes Salicornia virginica and Spartina value are normally interpreted to be indicative of shifts towards foliosa at 200, 400, and 700 mM NaC1 (equivalent to a range a greater reliance on CAM per se. This, presumably, is ac- of about 23 • 10 z kPa). As the sample size was not given and companied by the higher efficiency of water use by CAM plants few experimental details were provided, their results are difficult (Szarek and Ting, 1975) and could be of adaptive significance to interpret in the present context. Nevertheless, their data for to halophytes possessing this form of metabolism. Winter and Salicornia virginica represents a possible shift of 2.0%o. Admittvon Willert (1972) documented a clear induction of dark CO2 edly this seems insignificant, however, if Salieornia europaea ssp. uptake and malate accumulation in the halophyte Mesembryanthe- rubra behaves similarly, then, on the basis of our results (Fig. 5) mum crystallinum when plants were grown in the presence of NaC1. we would only expect a shift of about 2.2%o under their condiIn the absence of NaC1, CAM was not evident. Indeed, Bloom tions. Spartina foliosa, being a C 4 plant, might not be expected (1979) has shown that in this species, a significant degree of CAM to show a change in ~13C comparable to what we have observed cannot be induced by withholding water or by exposure to polyeth- in C3 halophytes. This would be particularly true if, in fact, the ylene glycol 6000. Equivalent osmolarities of NaC1, KC1, Na2SO4, effect of salt is to favour carbon fixation by PEP carboxyIase or KzSO4 are, however, all effective in promoting CAM (Bloom, over RuBP carboxylase, at least to some degree. Plants character1979; Winter, 1973). Stimulation of CAM by high NaC1 levels ized by the C4 pathway normally use PEP carboxylase as the primary enzyme of carboxylation (Hatch and Slack, 1970). results in a two-fold increase in water use efficiency of M. crystalliThe observation that 613C values are affected by O~a~ under num (Winter and Liittge, 1976). Treichel et al. (1974) found a dramatic increase in PEP carboxylase activity when Mesembryan- controlled conditions makes it unlikely that some factor other themum spp. were grown in 200 mM NaC1. Similar increases in than soil salinity is largely responsible for the differences in 613C PEP carboxylase activity were detected in Carpobrotus edulis but found along the transects. It is, however, important to note that isotopic fractionation by plants can be influenced by light intensity not in the halophytes Plantago maritima or Salicornia fruticosa. (Smith et al., 1976) and growth temperature (Smith et al., 1973; It was suggested that PEP carboxylase responds to NaC1 in halophytes of foreshore dune habitats but not in those typical of Smith et al., 1976; Troughton and Card, 1975), both of which salt marshes. Ganzmann and von Willert (1973) have reported undoubtedly change with transect position primarily as a result of changes in cover (Figs. 1 a and 2 a). the induction of CAM in the halophytic composite Aster tripolium. Smith et al. (1976) suggested that discrimination towards 13C Evidence for changes in photosynthetic patterns effected through salinization is not restricted to CAM plants. Shomer-Ilan by photosynthetically competent plants is at a minimum under and Waisel (1973) found that the C4 halophyte Aeluropus litoralis optimal environmental conditions. This was not the case in the present work where increased NaC1 concentrations resulted in showed CO2 fixation by the C3 pathway when salt was lacking. Sodium chloride also modified the balance between PEP carboxy- discrimination despite reduced growth. It is interesting to note lase and RuBP carboxylase in Zea mays and Chloris gayana, C 4 that in Salicornia (Fig. 4) very poor growth in the absence of NaC1 did not yield less negative ~13C values. glycophytes. Exposure to 25 mM and 50 mM NaC1 decreases the Within a single tissue, different classes of compounds may ratio between RuBP carboxylase and PEP carboxylase activities differ in 613C (Whelan et al., 1970; Smith and Jacobson, 1976). in wheat (Passera and Albuzio, 1978). A shift towards the "C4pattern" upon salinization has been shown in the C3 halophyte In particular, lipids can be considerably more depleted in 13C Plantago maritima (Ferron et al., 1977). Of interest are the effects than the rest of the plant (Smith and Epstein, 1970; Smith and of a saline environment on dark fixation of ~4CO2 by spinach Jacobson, 1976). Although it seems unlikely that the preferential accumulation of a particular compound in salt-stressed plants leaves (Joshi et at., 1962). Most of the label is found in amino acids (principally aspartic and glutamic acids), while organic acids could account for the large shifts in ~13C, it may be a contributing are favoured under non-saline conditions. Webb and Burley (1965) factor. The massive accumulation of proposed osmotica such as obtained similar results with Spartina alterniflora, a facultative betaine or proline in halophytes is a well documented phenomenon (Flowers et al., 1977). Shomer-Ilan et al. (1979) found that two halophyte. Dark fixation of ~CO2 by the halophytes Batis maritima, Borrichia frutescens, Salicornia europaea, and S. virginica species of lichen were more depleted in a3C during the winter results in most of the label being found in the amino acid fraction months. They suggested that this was due to the winter accumulation of 12C enriched storage materials and their subsequent disap(Webb and Burley, 1965). Few studies have been directed towards the influence of salt pearance during the summer. Differential fractionation of carbon isotopes by RuBP carboxyon stable carbon isotope ratios of higher plants. Smith and Epstein lase and PEP carboxylase is dependent upon the utilization of (1970) determined the ~13C values of nine salt marsh species but an essentially infinite source of CO2 (Berry and Troughton, 1974). did not look for shifts in these values within single species. In If the supply of COz to the site or sites of carboxylation in saltthe CAM plant Mesembryanthemum crystallinum, the 613C of total stressed halophytes was restricted relative to controls, then 6t3C tissue has been shown to change by 5.8~ after 3 weeks of treatvalues would become less negative, approaching that of the amment with 0.5 M NaCI (Osmond et al., 1973). Valiela et al. (t978) reported a very small difference of less than 1%0 between tall bient air. Salinization induces stomatal closure in the glycophyte Phaseolus to a degree which is inhibitory to photosynthesis (Gale and short growth forms of the C4 halophyte Spartina alterniflora. Recently, Maier and Kappen (1979), suspecting a possible in- et al., 1967; Longstreth and Nobel, 1979). Passera and Albuzio (1978) reported no differences in degree of stomatal opening befluence of CAM on variations of malate concentration as related to freezing tolerance, did not report any significant differences tween controls and salinized Triticum spp. In other glycophytes auch as Vitis (Downton, 1977) and Gossypium (Gale et al., 1967; in 6~3C values of controls and NaC1 treated Halimione portulacoides, a Ca halophyte. Card et al. (1974) appear to be the only Longstreth and Nobel, 1979), high salt concentrations reduce photoauthors who have specifically investigated the effect of salt on synthesis largely as a result of increased mesophyll resistance to 6~3C values of Ca or C4 plants. They reported no pronounced CO2 fixation. In halophytes the situation is more complex. Moder245

ate increases in salinity increase mesophyll conductance in Atriplex leucophylla, A. californica (De Jong, 1978) and A. halimus (Gale and Poljakoff-Mayber, 1970), while all levels of increasing salinity depress mesophyll conductance in Abronia maritima (De Jong, 1978). Mesophyll resistance is a measure of several combined nonstomatal factors, both physical and biochemical, which may limit photosynthesis. Therefore, it is difficult to assess or predict what effects increased mesophyll resistance might have on isotopic fractionation. Working with Spartina alterniflora, Longstreth and Strain (1977) hypothesized that photosynthetic response could be strongly influenced by salinity-induced changes in leaf structure but concluded that salinity rarely limits photosynthesis in this species. Although a shift in the balance between the relative activities of the two primary enzymes of CO2 fixation, RuBP carboxylase and PEP carboxylase, is, to us, the most attractive hypothesis to account for salinity-induced changes in 613C values of the C 3 halophytes Salicornia europaea ssp. rubra and Puccinellia nuttalliana, other explanations are possible and should be considered. Further investigations along these lines are now in progress.

Acknowledgements. We wish to thank Laura Boreiko and Nenita Lozano for technical assistance in isotope analysis. This research was supported by University of Calgary Thesis Research Grant TH 7829 to RDG, NSERC (Canada) grant A5257 to DMR, and NSERC (Canada) grant A8176 to HRK.

References

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