molecular, physiological, and growth responses to sodium stress in c4 ...

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E. WILLIAM HAMILTON, III,2,4 SAMUEL J. MCNAUGHTON,2 AND. JAMES S. .... a salt marsh species (Marcum and Murdoch, 1992; Naidoo and. Naidoo, 1998) ...
American Journal of Botany 88(7): 1258–1265. 2001.

MOLECULAR,

PHYSIOLOGICAL, AND GROWTH

RESPONSES TO SODIUM STRESS IN

C4

GRASSES FROM A

SOIL SALINITY GRADIENT IN THE

SERENGETI

ECOSYSTEM1

E. WILLIAM HAMILTON, III,2,4 SAMUEL J. MCNAUGHTON,2 JAMES S. COLEMAN3

AND

Biological Research Laboratories, Syracuse University, Syracuse, New York 13244-2012 USA and 3 Desert Research Institute, Reno, Nevada 89506 USA

2

The concentration of soil sodium (Na) is an important factor that influences species distribution in the Serengeti short-grass plains, Tanzania. Experiments were conducted to characterize physiological (growth, photosynthetic, nutrients, and water relations) and molecular (heat shock proteins and organic solutes) responses to high soil sodium in four Serengeti C4 grasses. The species tested were Andropogon greenwayi and three species of Sporobulus, S. ioclados, S. kentrophyllus and S. spicatus. Andropogon greenwayi occurs on locations with low soil Na concentrations, S. ioclados on low to moderate, S. kentrophyllus moderate to high, and S. spicatus on soils with high Na concentration. Among all four species, short-term physiological and molecular responses to Na treatments (0, 100, and 400 mmol/L Na) were correlated with their field soil Na concentrations. Sporobulus kentrophyllus and S. spicatus exhibited rapid molecular induction of heat shock proteins in response to experimental soil Na treatments within 24 h and had increased levels of proline within 96 h in contrast to A. greenwayi and S. ioclados. Photosynthetic rates and water relations were positively correlated with field soil Na concentrations and Hsp induction was clearly associated with photosynthetic tolerance. Long-term (6 wk) responses of the four species to Na treatment were consistent with the short-term responses to Na. Species that occur on low Na soils in the field did not survive past week 1 when treated with 400 mmol/L Na and exhibited significant reductions in biomass when treated with 100 mmol/L Na. Reduced biomass was associated with increased shoot tissue Na concentrations, and thus Na tolerance correlated with the Na concentrations of field leaf tissue. The results demonstrate that the community distribution of these species reflects their Na tolerance and that the observed physiological and molecular responses in tolerant species may have adaptive significance. Key words:

Andropogon greenwayi; heat shock proteins; Na stress; Serengeti; Sporobulus spp.

The Serengeti short-grass plains is a mosaic of grass species in which species distribution is greatly influenced by various environmental gradients, including rainfall, topography, and soil characteristics (Banyikwa, 1976; McNaughton, 1985). In particular, the concentration of soil sodium (Na) is correlated with topography (Banyikwa, 1976; McNaughton, 1985) that influences water runoff and therefore concentrates Na in low drainage basins. Among the dominant forage grasses of the southeastern Serengeti National Park, Tanzania, are three species of Sporobulus, S. ioclados, S. kentrophyllus and S. spicatus, which occupy different habitats on the plains (McNaughton, 1983a). Also abundant along the western edges of the Sporobolus grasslands is Andropogon greenwayi, an endemic species that forms virtual monocultures in a mosaic with other species at the landscape level (Banyikwa, 1976; McNaughton, 1985). Sporobulus kentrophyllus occurs on low flat basins with moderate to high soil Na concentrations and S. ioclados occurs on high and mid elevation sites with low to moderate soil Na concentrations (Banyikwa, 1976). Sporobulus spicatus occurs only on low-elevation drainage basins with high soil Na concentration, and A. greenwayi occurs on high Manuscript received 25 July 2000; revision accepted 5 December 2000. The authors thank M. McNaughton for invaluable technical assistance and S. A. Heckathorn and two anonymous reviewers for many useful comments on the manuscript. This research was supported by NSF (SJM) and Andrew W. Mellon (JSC). 4 Author for correspondence, current address: Washington and Lee University, Department of Biology, Lexington, VA 24450 (e-mail: hamilton@ wlu.edu). 1

elevation sites with low soil Na concentrations (Banyikwa, 1976). All of these species possess the C4 photosynthetic pathway. The relationship between the distribution of these species and soil Na suggests underlying differences in Na tolerance exist (Banyikwa, 1976). However, their relative tolerance to soil Na is unknown, as well as any physiological adaptations. A primary response of plants when exposed to increased soil Na is a decrease in plant water potential, because the osmotic and water potential of the soil decreases. One well-characterized response of plants to low soil water potential is the accumulation of osmotically compatible cellular solutes (e.g., proline and glycine betaine) as a mechanism to decrease plant osmotic and water potential. In native species, osmotically compatible solute accumulation is correlated with soil Na gradients and reduced physiological stress (Cavalieri and Huang, 1979; Briens and Larher, 1982; Hester, Mendelssohn, and McKee, 1996). In several species that accumulate proline, there is a delay (3–6 d) in proline accumulation following the onset of Na stress (Brady et al., 1984; Lerner, 1985; Yoshiba et al., 1997). Therefore, the early stages of Na stress are critical for plant survival because plants are not capable of osmotically adjusting water potential during this period. It is possible that prior to the accumulation of protective solutes heat shock proteins (Hsps) may play a protective role. Accumulating evidence suggests that Hsps play a role in tolerance to a variety of stresses (e.g., Downs, Ryan, and Heckathorn, 1999). Heat shock proteins are general stress proteins involved in maintaining cell function and thus survival during stress or facilitating recovery from stress (Vierling, 1991; Par-

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sell and Lindquist, 1994; Downs et al., 1997; Downs and Heckathorn, 1998; Guy and Li, 1998; Heckathorn et al., 1998). Of particular interest, because of their identified importance in many types of plant stress, are two classes of Hsps, the Hsp 70 class (70 kD) and the small Hsp (smHsp) class (15–30 kD) (Vierling, 1991; Parsell and Lindquist, 1994). The Hsp70 class of proteins are chaperones and are found in the cytosol and most organelles (Vierling, 1991; Wang, Goffreda, and Leustek, 1993; Parsell and Lindquist, 1994). The smHsp class consists of five major forms localized to the (1) cytosol (2 forms), (2) chloroplast, (3) mitochondria, and (4) endoplasmic reticulum. Recently, functions of the chloroplast and mitochondrion localized proteins have been identified. They both protect electron transport when plants are exposed to heat and oxidative stress (and also photoinhibition in chloroplasts; Downs and Heckathorn, 1998; Heckathorn et al., 1998; Downs, Ryan, and Heckathorn, 1999). Since salt stress increases cellular ionic concentration, which can affect protein synthesis and structure (Brady et al., 1984; Yoshiba et al., 1997; Hare, Cress, and Van Staden, 1998), the induction of Hsps by Na is likely to be an adaptive trait, which we predict is associated with Na-tolerant species. In addition to the cellular adaptations to salt stress, many species that are exposed to persistently high Na concentrations exude Na salts onto the surface of leaves (Lipschitz and Waisel, 1974; Drennan and Pammenter, 1982; Marcum and Murdoch, 1992; Marcum, Anderson, and Engelke, 1998; Naidoo and Naidoo, 1998). Exudation exists and is effective at reducing the concentration Na in tissues in Sporobulus virginicus, a salt marsh species (Marcum and Murdoch, 1992; Naidoo and Naidoo, 1998), and other Sporobulus species (Lipschitz and Waisel, 1974). Exudation of Na from the leaf allows for the uptake of water and nutrients while eliminating Na (Lipschitz and Waisel, 1974; Naidoo and Naidoo, 1998). To investigate the Na tolerance of these Serengeti species as well as important molecular and physiological responses related to Na stress/tolerance, two experiments were performed. The first experiment investigated short-term (4-d) molecular and physiological responses. Assays of content of Hsps and the osmotically compatible solute proline provided data at the molecular level, and measures of net photosynthesis and plant water status provided data that demonstrated plant stress or tolerance at the physiological level. The second experiment investigated long-term physiological and growth responses to Na treatment. Analysis of biomass accumulation and tissue concentrations of Na provided data on stress and/or tolerance at the whole-plant level. Collectively, the data link short-term molecular and physiological responses to long-term wholeplant responses that are correlated with the observed community distribution of these species in the Serengeti shortgrass plains. MATERIALS AND METHODS Analysis of field samples—Soil cores were collected in the Serengeti along with the corresponding leaf blade tissue at the beginning of the growing season and both were analyzed for Na concentration with an inductively coupled plasma spectrophotometer (Leeman Labs, Hudson, New Hampshire, USA) using protocols and standards as described in McNaughton (1988). Sample collection of each species occurred in sites where each species was dominant (S. J. McNaughton, personal observation). Growth chamber experiments—Plants were propagated vegetatively from greenhouse stocks maintained at Syracuse University in growth chambers sup-

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plying 650 mmol·m22 ·s21 of photosynthetically active radiation (PAR) set to a 12 h : 12 h day : night cycle with temperature programmed to a Serengeti daily average cycle (high : low, 328 : 158C; McNaughton, 1985). Andropogon greenwayi and S. ioclados growth occurs from basal tillers, and S. kentrophyllus and S. spicatus growth occurs from basal tillers and from stolons. The differences in growth form generated differences in biomass, but individuals were representative of their growth form in the field (Banyikwa, personal communication, Syracuse University, Syracuse, New York, USA). Plants received 100 mL of one-half strength Hoagland’s solution with Na added in equimolar portions of NaCl and NaHCO3 to 100 or 400 mmol/L Na. The Na treatment attempts to mimic the conditions in the short-grass plains just after the grasses break dormancy from the dry season, when early season rainfall should increase the Na concentration in the soil by bringing Na salts into solution. A preliminary experiment was conducted with S. ioclados, S. kentrophyllus, and S. spicatus using 50, 100, and 200 mmol/L Na treatments (data not shown). The results showed that the concentration at which biomass was most significantly reduced without plant death was 100 mmol/L for S. ioclados and 200 mmol/L for S. kentrophyllus, although 2 of 4 S. kentrophyllus replicates died at 200 mmol/L Na. Sporobulus spicatus biomass was not significantly reduced by any Na level and therefore the 400 mmol/L treatment was added. Proline was quantified in this experiment on subsamples and was not detected until 4 d after Na treatment. Photosynthetic measurements were made on the most recently fully expanded leaf from 1000 to 1100 using an open-flow gas-exchange system with a 2 3 6 cm leaf chamber (ADC, UK). Leaf chamber conditions were maintained at growth chamber conditions (650 mmol PAR 355 ppm CO2, 228C, and 50% relative humidity). Midday water potential (CW) was measured using a Scholander type pressure bomb (PMS Instrument Co., Corvallis, Oregon, USA) and solute potential (CS) using a dewpoint hygrometer (Wescor, Logan, Utah, USA). Experiment 1: Short-term responses—Plants (7–10 tillers) were grown in 10 cm width 3 10 cm height square pots. The experimental design was 4 treatments 3 4 species 3 3 replicates 3 2 harvests. Treatments consisted of three Na levels (0, 100, and 400 mmol/L) and heat shock at 0 mmol/L Na. Heat shock was performed at 438C by ramping the temperature from 258 to 438C over a 2-h period, as described in Heckathorn, Downs, and Coleman (1998b). Harvests were performed 24 and 96 h after treatment. Photosynthesis, CW, and CS were measured daily. Proline content was quantified using a modified method of Bates, Waldren, and Teare (1972) on day 4 in plants from the short-term experiment. Leaf tissue frozen in liquid N2 (0.1–0.2 g) was extracted in 2 mL of 3% sulfosalicylic acid for 5 min and centrifuged for 2 min at 10 000 rcf. One mL of supernatant was reacted with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid for 1 h at 1008C. The reaction was stopped by placing test tubes in an ice bath and then samples were extracted with 5 mL of toluene. The absorbance at 520 nm of the proline-containing organic phase was determined. Toluene served as a blank (Bates, Waldren, and Teare, 1972). Glycine-betaine is an important osmotic solute, and it would have been valuable to quantify, but the equipment required for analysis was not available. For protein extraction, plants were frozen in liquid N2 and stored at 2208C. Total leaf proteins were extracted and protein per unit leaf area was determined as described elsewhere (Downs et al., 1997; Heckathorn et al., 1998). Proteins (50 mg total) were separated by one-dimensional SDS-PAGE using 1 mm thick, 10 3 8 cm gels and then electroblotted to nitrocellulose membranes (Downs et al., 1997; Heckathorn, Downs, and Coleman, 1998). Membranes were individually probed with one of four antibodies: a polyclonal antibody to a DnaK-like chloroplast Hsp70 (Wang, Gofreeda, and Leustek, 1993), a commercial monoclonal Hsp70 antibody (SPA-820; StressGen, Vancouver, British Columbia, Canada), a polyclonal antibody that specifically reacts with the heat-shock domain of the small Hsps in plants (Downs et al., 1997), and a polyclonal antibody that specifically cross reacts with the methionine-rich domain of the small chloroplast heat-shock protein (Heckathorn et al., 1998). Proteins were visualized using a chemiluminescent detection system (Pierce Supersignal, Pierce Chemical Co., Rockford, Illinois, USA), and immunoblots were digitized with a Hewlett Packard IIs scanner (Hewlett

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Fig. 2. Grand mean 1 1 SE of all 4 d for net photosynthetic rate in A. greenwayi, S. ioclados, S. kentrophyllus, and S. spicatus plants from the shortterm experiment treated with 0, 100, or 400 mmol/L Na. Fig. 1. Mean 6 1 SE field leaf blade Na concentrations and mean 6 1 SE field soil Na concentrations (mg/kg dry mass) (circles; v) for Andropogon greenwayi, Sporobulus ioclados, S. kentrophyllus, and S. spicatus. Paired leaf and soil samples were collected in the Serengeti short-grass plains. Diamonds (l) represent experimental Na treatment concentrations.

Packard Co., Palo Alto, California, USA). The immunoblot data presented represent three replicates. Experiment 2: Long-term responses—Plants (7–10 tillers) were grown for 7 wk (1 wk of acclimation after transplanting) in polyvinylchloride tubes that were 30 cm height 3 6.35 cm diameter and in a mix of sand and perlite (3 : 1, volume : volume). The experimental design was 3 Na levels 3 4 species 3 3 or 4 replicates. Deaths resulting from transfer shock in S. ioclados and A. greenwayi allowed for only three replicates in each treatment. Plants received one-half strength Hoagland’s solution via cotton rope wicks inserted into a bottle attached to the pot (Marrone, 1982; Toft, McNaughton, and Georgiadis, 1987). The wicking of nutrient solution prevented dilution of Na treatments, which could occur by soil surface watering. A layer of perlite on the soil surface minimized evaporative loss of water. Photosynthesis was measured weekly. Midday water potential could only be measured on the first week after treatment because leaves tore in the pressure chamber on weeks 2 and 3 as a result of increasing succulence due to water stress. Plants were harvested after 6 wk and were separated into root and shoot tissue. Tissue was oven dried for 4 d at 508C and then weighed. Shoots were rinsed in 20 mL of double distilled water to remove any salts that may have been exuded. Rinse samples were analyzed for cation mineral elements by inductively coupled plasma spectrophotometry using the same protocols as mentioned previously. A commercial water-testing kit was used for Cl2 quantification (Lamotte, Chestertown, Maryland, USA). Mineral element concentrations were log10 transformed. Statistical analysis was performed using the ANOVA module of Statistica 5.0 (Statsoft, Inc., Tulsa, Oklahoma, USA). The death of all 400 mmol/L-treated plants in the long-term experiment, with the exception of S. spicatus, generated missing values in the ANOVAs. Therefore ANOVAs for all physiological variables were performed using only 0 and 100 mmol/L-treated plants. ANOVAs with all Na levels were performed separately on S. spicatus to determine treatment effects on physiological variables. Tukey post-hoc comparisons were performed to distinguish significant differences in interaction terms.

RESULTS Short-term Na responses—Analysis of field soil samples taken immediately below plants growing in the Serengeti, whose leaf tissues were also sampled, identified a clear gradient of soil Na concentration that was reflected in plant Na

content (Fig. 1). The photosynthetic activity of all species, except for S. spicatus, was significantly reduced by the addition of Na (Species 3 Na; P 5 0.0005; Fig. 2). Andropogon greenwayi and S. ioclados plants treated with 100 and 400 mmol/L Na exhibited 63 and 87% and 43 and 75% reductions in photosynthetic rate, respectively. The photosynthetic rate of S. kentrophyllus was unaffected by the 100 mmol/L Na treatment but was reduced by 50% in 400 mmol/L Na-treated plants. In Na-treated A. greenwayi and S. kentrophyllus plants, CW became gradually more negative by day 4 (Fig. 3). In Natreated S. ioclados plants, CW was highly negative at day 2 and this was maintained to day 4. In Na-treated S. spicatus plants, CW was more negative on day 1 and by days 3 and 4, 100 and 400 mmol/L-treated plants were not significantly different from 0 mmol/L Na-treated plants (Species 3 Na 3 Day; P , 0.0001). Solute potential (CS) was significantly different among species and more negative in all species when treated with Na, with the exception of S. spicatus (Species 3 Na treatment; P 5 0.0002; Fig. 4A). Proline content in 100 and 400 mmol/Ltreated plants was greater than in 0 mmol/L Na-treated plants, and S. kentrophyllus and S. spicatus accumulated greater quantities (Species 3 Na treatment; P , 0.0001; Fig. 4B). Na treatment increased leaf Na concentration in all species with significant differences in accumulation in each species (Species 3 Na treatment; 24 h, P , 0.0001 and 96 h, P , 0.0001; Fig. 5A, B). Andropogon greenwayi and S. kentrophyllus 400 mmol/L-treated plants accumulated significantly more Na than S. ioclados and S. spicatus. Andropogon greenwayi and S. ioclados did not exhibit increased production of either class of Hsp (Fig. 6; lanes 1–3, 5–6), although the production of Hsp 70 and members of the smHsp class were induced by heat-shock treatment (Fig. 6; lanes 4 and 8). Sporobulus kentrophyllus increased production of cpHsp70 and the cytosolic smHsps in response to the 100 mmol/L Na treatment, but not in response to the 400 mmol/L treatment (Fig. 6; lanes 9–12). Sporobulus spicatus increased production of cpHs70, Hsp70, three smHsps, and cpsmHsp in response to 100 and 400 mmol/L Na (Fig. 6; lanes 13–16). Additionally, S. spicatus produced a novel Hsp70 band in response to the 400 mmol/L Na treatment (Fig. 6; lane 15).

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Fig. 3. Mean 6 1 SE for midday water potential (CW; MPa) on days 1–4 for A. greenwayi, S. ioclados, S. kentrophyllus, and S. spicatus plants treated with 0, 100, or 400 mmol/L Na.

Long-term Na responses—Midday water potential in 400 mmol/L Na-treated plants could not be measured in A. greenwayi, S. ioclados, or S. kentrophyllus because all replicates had died within a week of Na treatment. All S. spicatus replicates survived the 400 mmol/L Na treatment. Midday water potential in 100 mmol/L Na-treated plants was significantly more negative in all species with the exception of S. spicatus (Species 3 Na treatment; P 5 0.0031; Table 1). Sporobulus spicatus was analyzed separately with all treatments included, and CW in 400 mmol/L-treated plants was 0.3 MPa more negative than in 0 and 100 mmol/L Na-treated plants (Na treatment; P 5 0.0025; Table 1). Sodium treatment of 100 mmol/ L reduced net photosynthesis in A. greenwayi, S. ioclados, and S. kentrophyllus throughout the experiment by 92, 38, and 20%, respectively (Species 3 Na treatment; P , 0.0001; Table 1). Net photosynthesis in S. spicatus was not affected by Na treatment when analyzed separately including 400 mmol/Ltreated plants (Na treatment; P 5 0.9124; Table 1). Total biomass was reduced in A. greenwayi, S. ioclados, and S. kentrophyllus by the 100 and 400 mmol/L Na treatments by 53 and 70%, 55 and 73%, and 30 and 71%, for each species, respectively (Species 3 Na treatment; P , 0.0001; Fig. 7). Total biomass in S. spicatus was not affected by Na treatment. Species differed in their shoot and root biomass responses to Na treatment. This is best demonstrated by the significant changes in root to shoot ratios (Species 3 Na treatment; P 5 0.0014; Table 1). Root to shoot ratios in A. greenwayi and S. ioclados increased with Na treatment that resulted from reductions in shoot mass without reductions in root mass. Na treatment increased root and shoot Na concentration in all species with significant differences in accumulation in each

species (Species 3 Na treatment; P , 0.0001). Andropogon greenwayi, S. ioclados, and S. kentrophyllus plants treated with 400 mmol/L Na accumulated significantly more Na than S. spicatus; the 100 mmol/L-treated plants accumulated similar concentrations of Na in all species (Table 1). Sodium treatment reduced root and shoot Ca concentration (Na; P , 0.0001; Table 1) but did not affect K concentrations (Na; P 5 0.69; Table 1). Rinse samples of leaf tissue that were collected to detect exudation contained Na and Cl2 (Table 1). Sodium treatment increased the concentration of Na and Cl2 in leafrinse samples but only in S. spicatus (Species 3 Na for Na and Cl; P , 0.0001). No other mineral elements (Ca, K, Mg, P) were detected at significant levels in sufficient replicates to warrant further analysis (data not shown). DISCUSSION To our knowledge, this is the first demonstrated induction of Hsps in response to soil Na and the first study relating Hsp production in naturally occurring plants to field soil Na levels in which each species grows. The data show that photosynthetic tolerance correlates with the observed levels of Na in field tissues, which also correlates with the molecular adaptation of Na-induced Hsp production. Long-term biomass responses were correlated with short- and long-term physiological responses in a direct relationship with the field Na gradient. In S. spicatus an additional mechanism for Na tolerance was the exudation of Na salts from leaves, and this reduced tissue concentrations to levels that were apparently tolerable for growth. The results support the hypothesis that the species

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have evolved species-specific tissue Na tolerances in relation to their field soil Na concentrations.

Fig. 4. Mean 6 1 SE leaf (A) solute potential (CS; MPa) and (B) proline content (mmol/g fresh tissue) on day 4 for A. greenwayi, S. ioclados, S. kentrophyllus, and S. spicatus plants from the short-term experiment treated with 0, 100, or 400 mmol/L Na.

Short-term molecular and physiological responses—The accumulation of leaf Na and proline in the four species was negatively correlated with the field concentrations of soil Na that these species experience. Interestingly, S. spicatus (very Na tolerant) and S. ioclados (Na sensitive) both accumulated relatively less leaf Na within 24 h of treatment. This suggests that S. ioclados and S. spicatus may not transport Na to shoots. The effect of partial exclusion of transport of Na to shoots was detrimental to photosynthesis and water relations for S. ioclados but did not effect S. spicatus (except for increased proline production). In contrast, S. kentrophyllus (moderately Na tolerant) and A. greenwayi (very Na sensitive) both exhibited higher concentrations of leaf Na at 400 mmol/L but exhibited lower concentrations at 100 mmol/L. Thus, Na tolerance in these grasses was not strictly related to salt exclusion. Proline production decreases the osmotic potential of cells, which can increase the uptake of water (Kramer and Boyer, 1995; Hare, Cress, and Van Staden, 1998). Additionally, increased levels of free proline are an indicator of protein degradation (Yoshiba et al., 1997). The role of proline may be serving a protective role in S. spicatus and S. kentrophyllus. In A. greenwayi and S. ioclados, proline accumulation is more than likely the outcome of protein degradation because photosynthetic Na tolerance was not demonstrated in these species and their water status did not recover from Na treatment. In Spartina species that grow along a marsh salinity gradient (Storey and Wyn Jones, 1978), and in agricultural species, proline production reduces water stress and is correlated with photosynthetic tolerance (Ladyman et al., 1983; Brady et al., 1984; Madan et al., 1994). In other species, a delay in the accumulation of proline in response to Na has been observed (Storey and Wyn Jones, 1978; Cavalieri and Huang, 1979; Briens and Larher, 1982; Madan et al., 1994). This delay can be attributed to the complex integration of stress signals that are required to generate a stress response (Hare, Cress, and Van Staden, 1998). In contrast to proline accumulation in these species, the Hsp response to Na treatments occurred rapidly. Within 24 h, S.

Fig. 5. Mean 1 1 SE leaf Na concentration (mg/kg dry mass) for A. greenwayi, S. ioclados, S. kentrophyllus, and S. spicatus at (A) 24 and (B) 96 h in plants from the short-term experiment treated with 0, 100, or 400 mmol/L Na.

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Fig. 6. Western immunoblots of whole-leaf extracts from plants in the short-term experiment subjected to either 0 (lanes 1, 5, 9, and 13), 100 (lanes 2, 6, 10, and 14), 400 (lanes 3, 7, 11, and 15) mmol/L soil Na, or whole-plant heat shock (lanes 4, 8, 12, and 16). Arrows to the right of blots correspond to molecular mass markers in kilodaltons (kD).

TABLE 1. Mean 6 1 SE for midday water potential, photosynthesis, root : shoot, shoot and root Na, Ca, and K, and rinse Na and Cl for Andropogon greenwayi, Sporobulus ioclados, S. kentrophyllus, and S. spicatus plants from the long-term experiment treated with 0, 100, or 400 mmol/L Na. Na

CW (MPa) Photosynthesis (mmol CO2·m22·s21) Root: Shoot Shoot Na (mg/kg) Root Na (mg/kg) Shoot Ca (mg/kg) Shoot K (mg/kg) Exuded Na (mg/kg) Exuded Cl2 (mg/kg)

0 100 400 0 100 400 0 100 400 0 100 400 0 100 400 0 100 400 0 100 400 0 100 400 0 100 400

A. greenwayi

21.07 6 0.04a 22.00 6 0.12b — 11.32 6 0.79a 0.86 6 0.34b — 0.69 6 0.1a 0.98 6 0.18a 1.23 6 0.06b 718 6 381a 7940 6 1433b 42203 6 2630c 648 6 120a 2677 6 596b 38133 6 5622c 3276 6 318a 3749 6 224a 4081 6 618b 19669 6 1651a 17902 6 716a 22487 6 3909a 220.6 6 14.5a 254.5 6 24.5a 218.1 6 32.5a 246.2 6 10.5a 285.2 6 28.2a 271.3 6 32.5a

S. ioclados

21.15 6 0.16a 21.82 6 0.06b — 10.09 6 0.97a 7.33 6 1.17b — 0.32 6 0.05a 0.71 6 0.20b 1.02 6 0.23b 2443 6 199a 5942 6 726b 16091 6 2630c 935 6 217a 5685 6 386b 41542 6 4142c 2954 6 134a 2468 6 14a 1894 6 240a 27495 6 2093a 27150 6 1007a 21185 6 1651a 270.5 6 38.5a 265.3 6 18.5a 303.4 6 27.8a 278.4 6 19.5a 296.2 6 24.6a 285.2 6 30.5a

S. kentrophyllus

21.12 6 0.08a 21.76 6 0.09b — 13.14 6 1.26a 10.53 6 0.92b — 0.73 6 0.01a 0.67 6 0.03a 0.60 6 0.03a 3268 6 181a 7404 6 407b 19555 6 4738c 628 6 124a 4937 6 461b 27860 6 8959c 2811 6 112a 2331 6 157a 2271 6 180a 18996 6 1534a 23790 6 800a 21613 6 1602a 259.8 6 25.5a 323.8 6 19a 297.7 6 16.8a 260.3 6 27.0a 272.8 6 24.8a 293.1 6 11.4a

S. spicatus

21.58 6 0.04a 21.59 6 0.03a 21.90 6 0.06a 14.31 6 1.58a 15.01 6 1.12a 14.19 6 1.29a 1.07 6 0.03a 1.01 6 0.05a 1.01 6 0.04a 2987 6 189a 7699 6 691b 7403 6 661b 752 6 201a 4726 6 638b 7193 6 459c 3136 6 91a 2457 6 287a 1668 6 168b 19115 6 177a 19756 6 1470a 16367 6 773a 520.3 6 184.4a 3925.3 6 671.2b 4820.9 6 1065.0b 258.4 6 25.5a 5397.6 6 596.8b 6160.7 6 567.2b

Note: Letters denote results of Tukey means comparisons within a species. Values with the same letter are not significant at P , 0.05.

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Fig. 7. Mean 1 1 SE total, root and shoot dry biomass for A. greenwayi, S. ioclados, S. kentrophyllus, and S. spicatus plants from the long-term experiment treated with either 0, 100, or 400 mmol/L.

spicatus and S. kentrophyllus produced chloroplast smHsps, other smHsps, and Hsp70. While each plant was subjected to Na-induced water stress, the production of cpsmHsps may protect photosynthetic proteins until water status can be balanced by osmotic adjustment. The cpsmHsp protects Photosystem II electron transport in response to heat, oxidative, and photoinhibitory stress (Heckathorn et al., 1998) and is induced by various stresses besides heat stress (Downs, Ryan, and Heckathorn, 1999). The role that the Hsp70s play in salt stress may be similar to their heat stress role, i.e., to prevent aggregation and misfolding of denatured proteins (Vierling, 1991; Parsell and Lindquist, 1994; Guy and Li, 1998). Sporobulus kentrophyllus and S. spicatus produced Hsps and proline at Na treatment levels that are within the range of field soil Na concentrations and photosynthetic rates were not reduced by Na in these species. This demonstrates that Hsp and proline production are traits that are associated with Na tolerance. Further analysis would be required to determine whether these two traits are indeed mechanisms of Na tolerance. The osmotic stress that occurs as a result of Na stress may explain the combination of these two traits in the Na tolerant species. An increase in ionic strength within a cell can have profound affects on protein synthesis and function (Brady et al., 1984; Paleg, Stewart, and Bradbeer, 1984; Yoshiba et al., 1997; Hare, Cress, and Van Staden, 1998). Interestingly, proline has been shown to directly stabilize enzymes (Paleg, Stewart, and Bradbeer, 1984; Rajendrakumar, Suryanarayana, and Reddy, 1997), suggesting that the accumulation of proline may not only act to adjust cellular osmotic potential. The coordination of smHsps and Hsp70s in stabilizing protein function in response to heat stress has been demonstrated (Forreiter, Kirschner, and Nover, 1997). This coordination may be crucial to Na tolerance prior to proline accumulation. This idea is supported by the lack of production of Hsps in A. greenwayi and S. ioclados and in the concomitant reduction in photosynthesis with Na treatment. Long-term physiological and growth responses—The longterm physiological results suggest that each species has a different level of photosynthetic tolerance to water stress. Further,

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the species that naturally grow on low-to-moderate concentrations of soil Na did not survive beyond week 1 when treated with the highest Na concentration. These species also exhibited reductions in biomass at 100 mmol/L Na that correlated with the concentrations of Na in which they grow in the field. Sporobulus spicatus, which grows on soils with high Na soil concentrations, survived all Na treatments and experienced a slight reduction in root mass when treated with 400 mmol/L Na. In a survey of 25 species of salt tolerant grasses, Glenn, (1987) found that tolerance to Na, when defined as growth, was associated with lower leaf relative water contents. This was attributed to tissue osmotic adjustment that created an osmotic gradient from the soil into the plant (Glenn, 1987). Exudation of Na salts through salt glands exists and is an effective method for reducing the concentration Na in tissues in Sporobulus virginicus, a salt marsh species (Marcum and Murdoch, 1992; Naidoo and Naidoo, 1998), and other Sporobulus species (Lipschitz and Waisel, 1974). Sporobulus spicatus is the only species that showed evidence of salt exudation. Low concentrations of Na and Cl2 were quantified in all other leaf rinse samples but the concentrations of Na and Cl2 did not increase with Na treatment. Sporobulus spicatus exuded higher concentrations of NaCl in response to Na treatment but it was not different between 100 and 400 mmol/L treatments. This reflects the saturating ability of the exudation mechanism, which has been demonstrated in other species (Lipschitz and Waisel, 1974; Drennan and Pammenter, 1982; Bradley and Morris, 1991; Naidoo and Naidoo, 1998). Presumably, the Na tissue concentrations of S. spicatus could be significantly higher if it did not possess the ability to exude NaCl. Adaptive significance—In contrast to salt-sensitive A. greenwayi and S. ioclados, the short-term molecular responses of Hsp production and proline accumulation were present in S. kentrophyllus and S. spicatus and each exhibited short- and long-term tolerance to Na treatments. The Na treatment levels at which the molecular responses were expressed in each species corresponded to the field soil Na concentrations in which they grow. The importance of the molecular responses may be to protect cellular functions prior to the initiation of other long-term tolerance mechanisms (e.g., Na exudation in S. spicatus). Proline and the smHsps protect proteins from unfolding during stress (Paleg, Stewart, and Bradbeer, 1984; Rajendrakumar, Suryanarayana, and Reddy, 1997; Heckathorn et al., 1998) and thus maintain function. The Hsp70 class of proteins prevent aggregation of unfolded proteins and aid in the refolding of denatured, yet still functional proteins (Guy and Li, 1998), and their production in the Na-tolerant species may provide for rapid recovery of cellular function following Na stress. The variation in Na tolerance in the four species is likely involved in regulating species distribution and in the maintenance of grass productivity in the Serengeti short-grass plains. The maintenance of productivity is beneficial to grazers as well as the grasses (McNaughton, 1976, 1983b, 1985). The variation in soil Na creates a mosaic of forage quality with respect to Na content that could benefit grazers. Grazing in the Serengeti increases plant-available soil nitrogen and Na (McNaughton, Banyikwa, and McNaughton, 1997), and the presence of species that can tolerate higher soil Na provides a means for grazer acquisition of an important mineral element in animal diets.

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