Summary

CO2 uptake was reduced at salinities above 150 meq 1^-1 in Scirpus and 300 meq 1^-1 in Spartina. In contrast Salicornia exhibited no inhibition of CO₂ uptake even at 450 meq 1^-1 salinity. Analysis of the responses to intercellular CO₂ partial pressures showed that the inhibition of photosynthesis by high salinity in both Spartina and Scirpus is primarily accounted for by reduced photosynthetic capacity of the mesophyll, and secondarily, by reduced leaf conductances.

Species differences in relative growth rate (RGR) almost exactly opposed the differences in photosynthetic rates; the highest RGR was found in Salicornia and the lowest in Spartina. This reversal is accounted for by the greater allocation to photosynthetic shoots in Salicornia, which more than compensated for the lower photosynthetic capacity per unit surface area. RGR was more sensitive to salinity than photosynthetic rate in all three species, but the same relative sensitivities held. For Scirpus, reduced leaf elongation rates and changes in allocation patterns account for the greater limitation by salinity of RGR than of photosynthesis, and may be a primary factor restricting productivity of this species in saline habitats.

Introduction

In the research reported here, we compared photosynthetic and growth responses to salinity of three marsh species: Spartina foliosa, Trin. a C₄ grass species, Salicornia virginica L., a succulent C₃ shrub species, and Scirpus robustus, Pursh. a C₃ sedge. The first two are common in the highly saline tidal marshes of San Francisco Bay with Spartina occupying the low marsh at elevations from about mean sea level to mean high tide level and Salicornia occurring in the higher marsh (Mahall and Park 1976a). Scirpus, in contrast, is more common in the brackish water marshes occurring further inland in the Sacramento River estuary (Atwater et al. 1980). At some sites, such as the Sonoma Creek Marsh at the northern extension of the San Francisco Bay system (Ustin et al. 1982), all three species occur together with Scirpus occupying an intermediate zone between the other two species.

The objectives of this research were to determine how these contrasting species differ in their photosynthetic responses to salinity and how these differences relate to growth responses. Analysis of the photosynthetic responses were obtained primarily by examining intercellular CO₂ partial pressure dependence curves for photosynthesis. These curves define a "demand function" (Raschke 1979) related to the capacities for carboxylation, electron transport, etc. in the leaf (Farquhar and Sharkey 1982) and a "supply function" determined primarily by stomatal conductance. This analysis allowed separation of the effects of salinity into stomatal and mesophyll components.

Materials and methods

Plants of Salicornia, Spartina, and Scirpus were regenerated from rhizome sections in sand irrigated with a modified Hoagland's solution and transplanted into 7 x 30 cm pots containing a perlite and sand mixture. The pots were subirrigated by partial immersion into tanks containing 26 1 of aerated nutrient solution. Each tank contained up to 15 plants. After the plants were established, the nutrient solutions were gradually salinized with an artificial sea salt mixture (Rile mix) up to 150, 300 or 450 meq 1^-1; the control treatment consisted of unsalinized Hoagland's solution. The solutions were changed every 2 weeks and the pots were leached regularly to insure uniform concentrations. All measurements were made after development of new leaves under the final salinity regimes.

Plants were grown in a greenhouse with supplemental light supplied by 1,500 W metal-halide arc lamps for 15 h per day. Photon flux densities (PFD) and air temperatures were recorded periodically using a LI-COR Inst., Inc. quantum sensor and copper-constantan thermocouples, respectively, connected to a Campbell Scientific CR21 Micrologger. On clear days, which predominated during the measurement period, photon fluxes were 1,300 m mol m^-2 s^-1 and the daily total 43 mol m^-2. On cloudy days the values were 750 m mol m^-2 s^-1 and 27 mol m^-2, respectively. Evaporative cooling kept daytime air temperatures between 27 and 32° C; night temperatures were 15 to 19° C.

Photosynthetic CO₂ and water vapor exchange rates were measured with an apparatus similar to that described by Pearcy (1977). A single attached leaf of Scirpus or Spartina or a branch of Salicornia was enclosed within a glass windowed circular brass chamber with a thermostatted water jacket in the base and the lid. A fan maximized the boundary layer conductances and heat exchange between the leaf or branch and the chamber. Leaf or branch temperatures were measured with 0.7 mm diameter copper-constantan thermocouples. Light was measured with a silicon cell mounted on the lid of the chamber. Air of known CO₂ concentration was supplied to the chamber by mixing CO₂ free air and 1% CO₂ from compressed air cylinders with calibrated metering valves or rotameters. Humidity was controlled by condensation to a known dewpoint in a thermostatted condenser. The flow rate was measured with either a mass flowmeter (Flow Technology, Inc.) or with a differential pressure transducer (Validyne Engineering, Inc.) mounted across a flow restrictor made from a scintered glass disc. CO₂ concentrations were determined with a differential infrared gas analyser (Beckman Inst., Inc. model 865 or Horiba, Inc. model VIA 500-R) and water vapor concentration with a solid-state relative humidity probe (Weathermeasure model HM-111P). Light was supplied by a 1,500 W metal-halide lamp, and intensities controlled with wire screen filters.

Leaf conductances to CO₂ (g'₁) were calculated from the simultaneous measurements of CO₂ and water vapor exchange using the equations given by Wong et al. (1978), which yield molar flux units for conductances. Mesophyll conductances to CO₂ (gem) were calculated from the initial slopes of curves of photosynthetic rate versus intercellular CO₂ partial pressure [p(CO₂)] as outlined by Jarvis (1971). Expression of intercellular CO₂ as a mole fraction gave molar flux units for g'm. The method outlined by Farquhar and Sharkey (1982) was used to calculate the relative limitation to CO₂ uptake imposed by g'₁. In this method the relative limitation, l_s, is calculated from:
 

where A_o equals the expected photosynthetic rate if g'₁ were infinitely high and A is the measured photosynthetic rate at the normal atmospheric CO₂ partial pressure (rate at 32.5± 1.0Pa). Since at an infinite g'₁ there would be no CO₂ partial pressure difference between the atmosphere and cell walls, values of A_o were determined from CO₂ dependence curves at 32.5 Pa intercellular p(CO₂). For Spartina, this required extrapolation of several of the curves since g'₁ decreased strongly at high CO₂ and kept intercellular p(CO₂) below 32.5 Pa.

Gas exchange rates and g'₁ were calculated on the basis of the surface area of one side of the leaf in Scirpus and Spartina and, so that the measurements were comparable, using half of the round shoot surface area in Salicornia.

Relative growth rates (RGR) were measured using seedlings selected for uniformity of size. Growth conditions in these experiments were the same as those used for the photosynthesis measurements. After the gradual salinization period, the plants were allowed to equilibrate for 7-10 days. Harvests of 4-5 plants each were then made at 7-10 day intervals and weights were determined after oven drying. RGR was then calculated for each harvest interval and the average was calculated to give an overall mean for the growth period. Ash contents were determined by combustion of dried tissue samples at 500° C for 8 h in a muffle furnace.

Results

The effects of the salinity treatments on the photosynthetic responses are best illustrated by the response curves to intercellular CO₂ partial pressure (Fig. 3). Arrows indicate the net CO₂ uptake and intercellular p(CO₂) at ambient p(CO₂). As is characteristic of C₄ plants, Spartina exhibited steep initial slopes of the response curves at low intercellular p(CO₂) and an abrupt transition to saturation at higher intercellular p(CO₂). The CO₂ compensation point was near zero for the plants grown at 0 meq 1^-1, as is typical of C₄ plants, but shifted to 3.5± 1.4 Pa at 450 meq l^-1. Salinity did not affect the initial slopes of the CO₂ response curves but did affect the CO₂-saturated photosynthetic capacity.

Both Scirpus and Salicornia (Fig. 3) exhibited CO₂ responses typical of C₃ plants, with high CO₂ compensation points and only a slight tendency for saturation at high intercellular p(CO₂). For Salicornia, the effect of salinity on the CO₂ response was relatively small and the curves differed principally in the degree of curvature at high intercellular p(CO₂) rather than in the initial slopes or CO₂ compensation points. In contrast, salinities above 150 meq1^-1 had a large effect on the CO₂ responses of Scirpus. The primary effect was a reduced initial slope of the CO₂ response curves and at 300 and 450 meq 1^-1 a slightly increased CO₂ compensation point.

Analysis of g'₁ and g'_m (Fig. 4) also shows that the effects of salinity differed considerably among the species. For Spartina, g'_m was greater than g'₁ and was not significantly affected by salinity, whereas g'₁ was strongly reduced at 450 meq 1^-1 However, l_s, the limitation actually imposed by g'₁ was quite small, ranging from 10 to 15% in 0 to 300 meq 1^-1 and increased to only 30% at 450 meq 1^-1 salinity treatments. Thus, while the decreased g'_l contributed to reduced photosynthetic rates of Spartina at 450 meq l^-1, lower photosynthetic capacity in the CO₂ saturated portion of the response curve was primarily responsible for the observed reduction in photosynthetic rate. The salinity-induced changes in g'₁ appear to act in concert with changes in the mesophyll limitations on CO₂ uptake, so that intercellular p(CO₂) remains at the transition from CO₂ limitation to CO₂ saturation.

The CO₂ response curves for Scirpus also indicated that the primary effect of salinity was on the mesophyll limitations on photosynthetic capacity. This was especially apparent at 450 meq l^-1, where photosynthetic rates at 32.5 Pa intercellular p(CO₂) were only 20% of those at 150 meq l^-1. The decrease in g'₁ closely paralleled photosynthetic capacity so that intercellular p(CO₂) remained relatively constant at 22 to 24 Pa. Calculation of l_s at each salinity showed that it increased from 27 to 40% from 0 to 300 meq l^-1 but decreased to only 19% at 450 meq l^-1.

Salicornia showed no consistent patterns of changes in photosynthetic rates, g'_m or g'₁ in response to salinity. Photosynthetic capacity in Salicornia was relatively independent of salinity over this range, increasing to slightly higher values at 450 meq l^-1 than at lower salt concentrations. Calculation of l_s showed that it was relatively constant at 15 to 17% at salinities up to 300 meq l^-1 but increased to 35 at 450 meq l^-1.

Substantial differences in the salinity dependence of RGR (Fig. 5) and final plant dry weights (Table 1) were also apparent. Maximum RGR was highest in Salicornia and lowest in Spartina, a relationship opposite that found for CO₂ uptake rates. For Salicornia, RGR was stimulated by 150 and 300 meq l^-1 and showed only a modest decline at 450 meq l^-1. The dried shoots of Salicornia consisted of 26% ash for plants grown at 0 meq l^-1, and about 41% ash for the plants grown at higher salinities. These differences in ash content account for 50 and 100% of the increase in final dry weights of the plants grown at 150 and 300 meq l^-1, respectively, as compared to those grown at 0 meq l^-1. At 150 meq l^-1, the remaining 50% of the increased final dry weight of Salicornia is accounted for by the increased RGR. Spartina also showed an increase in RGR and final dry weight at 150 meq l^-1, but higher salinities inhibited both quantities. At 450 meq l^-1, little growth occurred after the first two weeks and most seedlings died. Growth of Scirpus was inhibited at all salinities.

Comparison of the relative effects of salinity on RGR revealed a close parallel with its effects on photosynthesis, except that growth was always more sensitive. Thus, in Salicornia growth was inhibited above 300 meq l^-1, while photosynthetic rates were not affected. All levels of salinity inhibited growth in Scirpus, while photosynthesis was inhibited only at concentrations greater than 150meq l^-1. In Scirpus, the fact that growth was more sensitive to salinity than photosynthetic rate was probably due to increasing allocation to below-ground structures at high salinities, as indicated by the root/shoot ratios (Table 1), and to inhibition of photosynthetic surface area expansion. As shown in Fig. 6, both the rates of leaf elongation and the final leaf lengths in Scirpus were greatly reduced by salinity.

Discussion

The effects of salinity stress on CO₂ exchange are usually analyzed by simply comparing changes in g'_m and g'_l. In most halophytes investigated so far, g'_m is relatively independent of salinity, while g'_l is considerably reduced (Gale and Poljakoff-Mayber 1970; Longstreth and Strain 1977; De Jong 1978). The results shown in Fig. 4 indicate that Scirpus and Spartina exhibit this response. However, analysis of the limitations from the CO₂ dependence curves following the approach of Farquhar and Sharkey (1982) shows that both stomatal and mesophyll limitations contribute to the reduced photosynthetic performance at high salinities. Where salinities are sufficient to strongly limit CO₂ uptake, the primary effect appears to be on the mesophyll. In Spartina, the changes in CO₂-saturated photosynthetic rates and the CO₂ compensation point, photosynthetic components which are not included in the calculation of g'_m, appear to account for nearly all of the changes in photosynthetic rate which occur in response to salinity. Thus, the effects of salinity on photosynthesis in Spartina are probably at the metabolic rather than the diffusional level. In Scirpus too, the CO₂ response curves show that salinity has a major effect on the limitations to CO₂ uptake in the mesophyll but the stomata, however, also play an important role in the responses to salinity, since l_s increases with increasing salinity up to 300 meq 1^-1 However, at 450 meq 1^-1, the reduction in photosynthetic rate due to lower g'_l was actually less than at other salinities because of the low inherent photosynthetic capacities of the mesophyll. Among these species, only Salicornia, the most halophytic species, shows no inhibition in photosynthetic rate or g'_m and little change in g'_l with increasing salinity.

The increase in the CO₂ compensation point of Spartina at high salinity suggests either a large stimulation of respiration or some breakdown in the C₄ mechanism. Giurgevich and Dunn (1979) reported high CO₂ compensation points under field conditions in a Georgia salt marsh for the short form of Spartina alterniflora, whereas the tall form, growing in less saline sites, had low compensation points. While salinity can stimulate respiration (Epstein 1972), it is unlikely that the increase could be large enough to cause the shift in the CO₂ compensation point observed in the plants grown at 450 meq 1^-1. The metabolic effect may be localized in the bundle sheath cells, since the RuBP carboxylase-oxygenase reactions potentially determining a compensation point and determining the CO₂ saturated photosynthetic capacity are localized there (Berry and Farquhar 1978). The lack of a salinity effect on the initial slope (g'_m) of the CO₂ response curves, which is presumably controlled by PEP carboxylase activities in the mesophyll cells, is consistent with this hypothesis.

RGR is ultimately a function of total photosynthetic capacity as determined by leaf photosynthetic rate and the proportional allocation to photosynthetic versus non-photosynthetic tissues. Comparisons of photosynthetic and growth responses to salinity among the three species clearly illustrate the importance of differences in allocation patterns, especially since photosynthetic rates and RGR were inversely related. Low RGR in Spartina, despite the high photosynthetic rates, was due to the low allocation to photosynthetic tissue as compared to the other species. The large allocation to stems in Spartina is probably necessary to keep the leaves above water level as much as possible and to resist mechanical damage during tidal flooding. Similarly, the high RGR of Salicornia can be accounted for by high allocation to photosynthetic tissue, which more than compensates for the low photosynthetic rates.

Comparisons within each species show that growth was always more sensitive to salinity than was photosynthesis, suggesting that photosynthesis, while certainly contributing to the growth response, is not the primary factor determining RGR in these species. These patterns of growth are consistent with the responses of other halophytes where comparisons have been made (Gale and Poljakoff-Mayber 1970; De Jong 1978; Winter 1979).

The differences in physiological and growth responses found in the three species correlate well with differences in field distribution and behavior. Midsummer salinities are much higher in the high marsh where Salicornia occurs than in the low marsh where Spartina is found (Mahall and Park 1976a; Ustin et al. 1982). Mahall and Park (1976c) suggest that Spartina is excluded from the high marsh by salinity while Salicornia is excluded from the low marsh by tidal flooding effects on seedling survival. Furthermore, they suggest that both species are less abundant in the intermediate zone because both high salinities and tidal flooding occur there. Our results demonstrate that Salicornia is much more tolerant of high salinities than Spartina, in terms of both growth and photosynthesis. The occurrence of Scirpus in the ecotone between Spartina and Salicornia is an intriguing paradox, since its photosynthetic and growth responses are more sensitive to moderate salinity levels than those of either Salicornia or Spartina. Indeed, midsummer salinities in the Scirpus zone are sufficient to inhibit strongly growth and photosynthesis of Scirpus. Field measurements have shown that g'_l values for Scirpus are quite low during the summer, supporting the view that photosynthetic gas exchange may be strongly limited (Ustin et al. 1982). However, these marshes have high salinities only during the summer; spring salinities are much lower because of higher fresh-water runoff into San Francisco Bay and leaching in the marsh due to the heavy winter precipitation. Scirpus completes vegetative growth during this spring period of lower salinities, whereas Spartina and especially Salicornia are more active during the late spring and summer (Ustin et al. 1982). The rapid growth of Scirpus at low salinities and the high photosynthetic rates at low temperatures would appear to favor this kind of seasonal growth pattern and allow survival of Scirpus in these marshes.

Acknowledgements

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