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Scientific
Publications - Work Done by Microbiology Reader
Plant and Soil, 260 (1-2): 237-251, March 2004
Evaluation of the roles of two compatible solutes, glycine betaine and
trehalose, for the Acacia senegal-Sinorhizobium symbiosis exposed to
drought stress
Leena A. Räsänen, Salla Saijets, Kari Jokinen and Kristina
Lindström ABSTRACT Acacia senegal (Mimosoideae) is a leguminous, nitrogen-fixing tree that grows in arid areas of Africa and the Near East. In this work, we studied the effects of drought stress on the development of symbiosis between A. senegal seedlings and Sinorhizobium arboris. We also evaluated if two exogenous compatible solutes, glycine betaine and trehalose, are advantageous for the A. senegal-Sinorhizobium symbiosis and if these solutes are capable of protecting two Sinorhizobium strains from salt stress (NaCl) and osmotic stress induced by polyethylene glycol (PEG 6000). A. senegal seedlings exposed to severe drought stress developed more root hairs than plants grown under moderate stress. After inoculationwith the GUSmarked S. arboris strain HAMBI 2180 the hairs were deformed but infection threads occurred only occasionally. Non-typically deformed hairs were dwarfed and swollen. Severely stressed roots contained less nodules but more nodule initials than moderately stressed ones. Nodules formed showed a lowered glucuronidase activity and signs of premature senescence. The numbers of culturable rhizobia in soil mix were reduced from 107 to 106 CFU g−1. Thirty days after inoculation severely drought-stressed A. senegal seedlings were wilted. Regarding endogenous glycine betaine, A. senegal appeared to be a non-accumulator plant but was able to translocate foliar-applied glycine betaine into roots. Both glycine betaine (0.01 M) and trehalose (0.01, 0.05 and 0.09 M) protected cell cultures of the wild type S. arboris strain HAMBI 1552 and S. saheli strain HAMBI 1496 from osmotic stress (9 and 17% PEG). In the case of salt stress only trehalose had a favourable effect. Application of 0.0003 M glycine betaine or trehalose into A. senegal soil mix exposed to severe drought stress maintained the numbers of culturable rhizobia at the same level as in moderately stressed soils. The presence of glycine betaine in the soil mix also helped A. senegal seedlings survive under severe drought. Abbreviations: BD – minimal Brown and Dilworth medium (1975); CFU – colony forming unit; EPS – exopolysaccharide; GB – glycine betaine; GUS – β-glucuronidase; MS – moderate drought stress; NAGGN – N-acetylglutaminylglutamine amide; PEG – polyethylene glycol; SS – severe drought stress; Tre – trehalose; VBNC – viable but nonculturable;WHC – water holding capacity; YEM – yeast-mannitol.
INTRODUCTION When water deficiency causes a decrease in soil or plant water potential, the plant can maintain turgor or reduce the rate of turgor loss by actively accumulating inorganic solutes. Under salt stress osmotic adjustment can occur through compartmentation of toxic ions from the cytoplasm into the vacuole (Hasegawa et al., 2000; Turner et al., 2000). Plants can also protect themselves from drought and salt stress by accumulating organic compounds called compatible solutes. Compatible solutes are non-toxic, highly soluble and uncharged molecules, which do not inhibit normal metabolic reactions but stabilise proteins and membranes (Csonka, 1989; McNeil et al., 1999). Compatible solutes also protect enzymes from heat inactivation (Paleg et al., 1981, 1984). Similar protection mechanisms are also found in bacteria. Under salt stress halobacteria maintain osmotic equilibrium by allowing influx of salt. Other bacteria exclude salt via the production of compatible solutes or other endogenous osmolytes (Galinski, 1995). For example, Sinorhizobium meliloti cells grown in NaCl can accumulate K+-ions and glutamate, and synthesise the following compatible solutes: N-acetylglutaminylglutamine amide (NAGGN), trehalose, and glycine betaine when its precursor, choline, is present in the growth medium (Smith et al., 1988; Miller andWood, 1996). If organic osmolytes are present in the surrounding medium, bacteria prefer uptake to synthesis de novo. Exogenous osmolytes that improve cell growth under adverse osmotic conditions are referred to as osmoprotectants (Galinski, 1995). Glycine betaine (N,N,N-trimethylglycine) is a preferred compatible solute for the majority of prokaryotes, and perhaps the most widely utilised osmolyte in the plant and animal kingdoms. Halophilic and halotolerant methanogens belonging to the Archaea and halotolerant oxygenic and unoxygenic phototrophic bacteria are capable of synthesising glycine betaine. Other bacteria convert glycine betaine from choline in a two-step enzymatic reaction or transport it directly from the environment (Galinski, 1995; Sleator and Hill, 2001). Glycine betaine is probably released into the soil by microbial producers upon dilution stress (rainfall, flooding), by decaying plant and animals, or /and by mammals in the form of excretion fluids (e.g., urine; Sleator and Hill, 2001). Choline is a common constituent of the eukaryotic membranes (phosphatidylcholine) and should therefore be widespread in the soil (Boncompagni et al., 1999). Plant polysaccharides, cellulose and starch, are presumably natural sources of disaccharide osmoprotectants (Gouffi et al., 1999). However, it is unknown to what extent osmoprotectants of soils are available for bacteria. Halotolerant plants synthesise and accumulate glycine betaine in the cytoplasm (e.g., in chloroplasts; Rhodes and Hanson, 1993). Also exogenously applied glycine betaine, by spraying to the foliage or adding into soil, was shown to improve drought tolerance of those kind of cultivated plants that lack or have low ability to accumulate glycine betaine (Agboma et al., 1997; Xing and Rajashekar, 1999; Diaz-Zorita et al., 2001). Acacia senegal is a leguminous tree, distributed in arid and semiarid areas of Africa and the Middle East. A. senegal has several properties that make it suitable for agroforestry and forestation of degraded dry areas: ability to fix nitrogen, drought tolerance and multipurpose uses (e.g., wood, fodder, gum arabic). Two extensively studied Sinorhizobium strains, the salt tolerant S. arboris strain HAMBI 1552 and the salt sensitive but heat tolerant S. saheli strain HAMBI 1496 (Zhang et al., 1992; Zahran et al., 1994), are capable of inducing nitrogen-fixing nodules on several African acacias (e.g., A. senegal), Afro-Asian P. cineraria and Latin-American Prosopis species (Räsänen et al., 2001). The two Sinorhizobium strains are potential inoculant rhizobia, showing good symbiotic performance and competitive abilities (Zhang et al., 1992; Räsänen et al., 2001). Drought stress is considered the major factor causing mortality among tree seedlings growing in arid and semiarid regions of the tropics (Khurana and Singh, 2001). In this work we studied how the water deficiency affects the symbiosis between A. senegal seedlings and Sinorhizobium sp. (i.e., growth of the partners, infection process and nodulation). To understand the role of compatible solutes for rhizobia, we investigated if the most common compatible solute, glycine betaine, can function as osmoprotectant for two Sinorhizobium strains exposed to salt and osmotic stress. We also evaluated if the addition of glycine betaine into the soil protects the development of the A. senegal-Sinorhizobium symbiosis from drought stress. Glycine betaine used in our study was extracted from sugar beet (Beta vulgaris). Another compatible solute, trehalose, was included in our experiments because it is a general stress response compound in microorganisms, playing an important role during desiccation (Potts, 1994). Some rhizobia, such as S. meliloti, can use trehalose present in surrounding medium for osmoprotection (Gouffi et al., 1999). Our work is the first study in which the influence of glycine betaine and trehalose was compared to each other and their roles were studied both from bacterial and plant viewpoint.
MATERIAL AND METHODS Bacterial strains Three Sudanese Sinorhizobium strains, S. arboris strain HAMBI 1552 isolated from Prosopis chilensis nodules, S. saheli strain HAMBI 1496 isolated from Acacia senegal nodules (Zhang et al., 1991; Nick et al., 1999) and S. arboris strain HAMBI 2180 were used in this study. Strain 2180 is a gusA gene marked derivative of strain 1552 (Räsänen and Lindström, 1999), containing the transposon mTn5SSgus21 carrying the gusA gene (Wilson et al., 1995). Rhizobia were grown at 28 °C on yeast extract-mannitol (YEM) agar with Congo red (Räsänen and Lindström, 1999) supplemented with streptomycin (250 µg mL−1).
Growth tests for bacteria In all stress experiments sinorhizobia were grown in minimal Brown and Dilworth (BD) medium (0.7 g KNO3, 0.25 g MgSO4 ×7H2O, 0.02 g CaCl2, 0.2 g NaCl, 0.36 g KH2PO4, 1.4 g K2HPO4, 6.6mg FeCl3, 0.15 mg EDTA, 1 mL vitamin solution [10 mg nicotine acid, 10 mg thiamine-HCl, 10 mg biotin, 10 mg Ca-panthotenate, 100 mL H20] per 1 L H20; Brown and Dilworth, 1975). To test appropriate glucose concentrations, rhizobial cells were grown in Erlenmeyer flasks containing 250 mL of BD broth for 5 days at 28 °C with shaking (150 rpm). For the determination of growth curves, optical densities were recorded with a spectrophotometer (A600nm) twice a day and cell numbers as colony forming units (CFU) by plate counts on YEM agar once a day. To test the effect of exogenous glycine betaine and trehalose on the growth of sinorhizobia under stress, bacteria were incubated in BD medium on multiwell plates in Bioscreen (Labsystems, Helsinki, Finland) for 5 days with shaking. The optical density (wide band filter) was continuously recorded (every two hours). Bioscreen wells were inoculated by adding 20 µL of fresh culture, which was diluted with sterile water to achieve the optical density of 0.7 Abs (A600nm), into 300 µL of growth medium. 0.1, 0.5 and 1.0 M NaCl and 9, 17 and 24% PEG 6000 (w/v) were used as stress substances. 0.01, 0.05 and 0.1 M glycine betaine (Finnsugar Bioproducts, Finland) and 0.01, 0.05 and 0.09 M trehalose (Fluka, BioChemika) were used as osmoprotectants. Five replicate wells were used for each combination. Some experiments were repeated by growing strains 1552 and 2180 in Erlenmeyer flasks containing 100 mL of BD broth, with sampling for optical density measurements and plate counts 0, 1, 4 and 8 days after inoculation.
Growing of plants A. senegal (L.) Willd. seeds collected from El Nuhud, Sudan were scarified and surface sterilised with concentrated H2SO4 and germinated as previously described (Räsänen and Lindström, 1999). The seedlings were grown in glass jars filled with 700 mL (930 g) of a sterile mixture of washed sand, vermiculite and ceramic Leca-gravel® (Räsänen and Lindström, 1999). After sterilisation, the volume of the soil mix in each jar was adjusted to 700 mL. Six germinated seeds were planted in each jar, and the seedlings were inoculated at the time of planting. For inoculation, cultures were grown in YEM broth to early stationary phase and diluted with sterile water. The seedlings were grown in a growth cabinet with an 18-h light period at 30 °C and a 6-h dark period at 21 °C. The plants were watered twice a week alternatively with sterile, nitrogen-free quarter strength Jensen’smedium (Vincent, 1970) or with sterile water.
Determination of glycine betaine A. senegal seedlings, originating from El-Nuhud and Gedarif in Sudan, were grown as described previously (Räsänen and Lindström, 1999), except that three germinated seeds per jar were sown and jars inoculated with 5 mL of bacterial solution (108 CFU mL−1). Six weeks after inoculation shoots were sprayed with 15 mL of 0.1 M glycine betaine in 0.1% Tween 20 (v/v; Mäkelä et al., 1996). The soil mix was covered with aluminiumfoil in order to prevent glycine betaine from being absorbed by the soil. Control plants were sprayed with 15 mL of 0.1% Tween 20. Seedlings were sampled 2 and 7 days after spraying. Plants were rinsed with sterile water and dried on the paper towel. Shoots and roots were separated, placed into 50-mL plastic tubes, frozen with liquid nitrogen and stored at −20 °C. Both treated and untreated samples were analysed for glycine betaine by liquid chromatography (HPLC) by using a cation exchange (Ca2+) column (Rajakylä and Paloposki, 1983).
Drought stress experiments The water holding capacity (WHC) of the soil mix (sand:vermiculite: Leca-gravel; 5:5:3) was 39.2%. The drought experiment included six treatments: (i) moderate drought stress (MS), (ii) MS with glycine betaine (GB), (iii) MS with trehalose, (iv) severe drought stress (SS), (v) SS with GB, and (vi) SS with trehalose. Before sowing, all jars were watered with 30 mL of Jensen solution. For inoculation, cultures of S. arboris strain 2180 grown in YEM broths to early stationary phase were diluted with sterile water at 107 CFU ml−1. The seedlings were inoculated at the time of sowing with 10 mL of this solution, by distributing it evenly on seeds and soil mix. 20 mL of 0.01 M glycine betaine or trehalose was added to GB or trehalose jars two days after sowing. At the same time, all jars were watered with 20 mL of water. At the set-up of the experiment, the soils contained approximately 0.0003Mglycine betaine or trehalose, and the bacterial density was approximately 4×105 CFU g−1. Watering was started four days after inoculation. Moderately stressed jars were watered with 30 mL (10% of WHC) and severely stressed jars were watered with 7.5 mL (2.2% of WHC) of Jensen solution or water. The plantswere grown for 42 days. The jars were watered twice a week. However, 30 days after inoculation severely stressed jars were so dry that all seedlings grown without compatible solutes were wilted. Therefore, all severely stressed plants were watered during the last week of the experiment every second day with 15 mL of water or quarter strength Jensen’s medium. Plant and soil samples were taken 10, 15, 21, 28 and 42 days after inoculation. Sampling was conducted three to four days after the watering. There were duplicate jars per each treatment and per each sampling day. The following parameters were determined: the numbers of plants survived, fresh weights and length of shoots, the numbers of nodules and their initials, CFU counts and soil moisture.
Determination of bacterial numbers and soil moisture Six soil samples (2–3 g) per jar were collected by using a 6 mm-wide and 30 cm-long corer. Subsamples were combined and mixed well. For CFU counts, duplicate samples of 2.0 g of soil mix were incubated with 18 mL of sterile Tween buffer (0.05Mphosphate, pH 6.5, 0.1% Tween 80) for 2 h at 28 °C with shaking (150 rpm). After incubation, the soil suspension was diluted with Tween buffer and plated on YEM agar containing Congo red. The numbers of bacteria were calculated as CFU per g of dried soil. The moisture content was determined by weighing duplicate samples of 10.0 g of fresh soil mix and drying them at 100 °C to constant weight.
Staining of roots and nodules for microscopy Root systems were incubated in modified GUS assay buffer (Wilson et al., 1995; Räsänen and Lindström, 1999) containing X-Glc-A 100 µg mL−1 for 3 days at room temperature with mild shaking. Roots, root hairs and nodules were observed under bright field microscopy and/or stereomicroscopy. Root systems were stored in GUS assay buffer at 4 °C.
Statistical methods Comparisons between treatments were carried out by using either one-way ANOVA or a non-parametric Kruskal-Wallis test to compensate for non-normal distribution of data and/or heterogeneity of variance. Tukey’s test was applied after ANOVA and t -test, in which equal variances were not assumed, was applied after the Kruskal-Wallis test to compare means atP < 0.05. All statistical analyses were performed with the program SPSS 10.0 for Windows.
RESULTS Growth of sinorhizobia with different carbon sources Glucose. In bacterial stress experiments, sinorhizobia were generally grown in Bioscreen in BD medium containing 1.0 g L−1 (0.006 M) glucose as a carbon source. When S. arboris strain 1552 was cultured in Erlenmeyer flasks for four days, the optical density was considerably higher with 2 g L−1 (0.011 M) glucose (approximately A600nm 1.0) compared with 0.006 M glucose (approximately A600nm 0.5). However, the cell numbers increased similarly at both glucose concentrations, being 5×108–109 CFU mL−1 at the end of incubation (Figure 1). This indicated that in 0.011M glucose rhizobial cells used the excess sugar for the production of exopolysaccharides (EPS).
Glycine betaine. From glycine betaine concentrations tested (0.01, 0.05 and 0.1 M), S. arboris 1552 and S. saheli 1496 strains could grow best when 0.01 M glycine betaine was used as a sole carbon source, although their growth showed some delay compared with the growth in 0.006 M glucose (Figures 2a, 3a). The presence of glucose together with glycine betaine did not benefit strain 1552 (Figure 2b) but slightly favoured strain 1496 (Figure 2a).
Trehalose. All concentrations of trehalose, i.e., 0.01, 0.05, 0.09 M, tested in Bioscreen with or without glucose, caused a substantial increase in the optical density of the sinorhizobial cultures (Figures 2b, 3a). Some experiments were repeated by growing strain 1552 and its GUS marked derivative 2180 in Erlenmeyer flasks. It appeared that with 0.01 M trehalose the cell numbers increased similarly as with 0.01 M glycine betaine (from 107 to 109 CFU mL−1, data not shown). However, optical densities were considerable higher with trehalose (A600nm 1.5–1.8) than with glycine betaine (A600nm 0.5–1.0). This suggested that in addition to cell growth sinorhizobia utilised trehalose for the production of EPS. Nevertheless, it was not known, which part of the optical density was caused by the increase in cell numbers and which part was due to the enhanced EPS production. Thus, the term growth used in the following text actually describes both cell proliferation and EPS production.
Effects of compatible solutes on cells exposed to salt and osmotic stress In cell culture experiments, salt stress was generated by using 0.1, 0.5 and 1.0 M NaCl. Osmotic stress was generated by using 9, 17 and 24% PEG 6000. PEG is a non-penetrating osmotic agent that is thought to restrict growth of bacteria due to the increase in solute concentration outside the cell. PEG may also cause matric stress, because PEG presumably reduces availability of water by binding water molecules. Generally, the growth of bacteria with compatible solutes, stress substances or their combination was compared with that in the basic minimal BD medium containing 0.006 M glucose. Depending on the stress substance (NaCl, PEG) and/or compatible solute (glycine betaine, trehalose) applied, there were big differences in the length and timing of the lag and exponential phases and in the final values of the optical densities. In addition, slow increase in optical density at start did not necessary implicate that the culture also later showed a poor growth. In general, use of trehalose as an osmoprotectant produced the best growth responses, i.e., the cultures had early and short exponential phases and high optical density values at the end of incubation. S. arboris strain 2180, which was the GUS marked derivative of strain 1552 and used in plant experiments, grew similarly to its parental strain (data not shown).
Salt stress. 0.1 M NaCl did not disturb growth of the salt tolerant strain 1552 and 0.5 M NaCl impaired its growth only slightly (Figure 2b). In the case of the salt-sensitive strain 1496 the results were contradictory because 0.1 M NaCl favoured and 0.5 M NaCl suppressed its growth (Figure 3b).
Compatible solutes under salt stress. At 0.5MNaCl, 0.01 M trehalose improved growth of the slightly salt stressed strain 1552 (data not shown) but this improvement was weak for the salt stressed strain 1496 (Figure 3b). Glycine betaine had no clear effect on sinorhizobia grown in 0.5 M NaCl (data not shown). From all tested conditions, the combinations of 0.1 M NaCl+0.01 M trehalose and 0.1 M NaCl+0.01 or 0.05 M glycine betaine caused the best growth responses (Figure 3b).
Osmotic stress. 9 or 17% PEG delayed growth of sinorhizobia. Strain 1496 was similarly impaired by 9 and 17% PEG, whereas strain 1552 grew better in 9% than in 17% PEG (Figures 2c, 3c.) Like 1.0 M NaCl (Figure 2a), 24% PEG inhibited growth of both strains almost completely (Figure 2c).
Compatible solutes under osmotic stress. All glycine betaine concentrations tested (0.01, 0.05 and 0.1 M) increased growth of strain 1552 exposed to osmotic stress (9 and 17% PEG; Figure 2c). 0.05 M glycine betaine produced the best results for strain 1496 grown in 9% PEG. In 17% PEG, the growth improvement was delayed (Figure 3c). Thus, both glycine betaine and trehalose functioned as osmoprotectants for sinorhizobia exposed to osmotic stress. However, the growth improvementwas more obvious with trehalose than with glycine betaine (Figures 2c, 2d, 3c).
Endogenous and exogenous glycine betaine on A. senegal The two Sudanese A. senegal provenances investigated, El Nuhud and Gedarif, contained only minor amounts of endogenous glycine betaine, for the shoots 13 (SE ±10) nmol g−1 dry wt and for the roots <3 nmol g−1 dry wt. A. senegal seedlings could absorb and translocate foliar-applied glycine betaine into root systems. Two days after treatment, the shoots of the provenance from El Nuhud contained 320 (SE ±69) nmol g−1 dry wt glycine betaine and five days later 190 (SE ±111) nmol g−1 dry wt. The values for the roots were 30 (SE ±32) and 80 (SE ±67) nmol g−1 dry wt, respectively. Also the other provenance from Gedarif could absorb glycine betaine. Seven days after spraying, the glycine betaine content in the shoots was 140 (SE ±2) nmol g−1 dry wt and in the roots 30 (SE ±15) nmol g−1 dry wt. However, the A. senegal foliage appeared to be sensitive to 0.01, 0.1 and 0.5 M glycine betaine, causing defoliation within a week after the treatment. Nevertheless, this was not an irreversible event and four weeks later new leaves had developed, and seedlings had a vigorous appearance. 0.001 M glycine betaine, 0.1% Tween 20 or sterile water did not cause any defoliation.
Inoculated A. senegal seedlings under drought stress When inoculated A. senegal seedlings were exposed to moderate and severe drought stresses, the numbers of culturable rhizobia were significantly lower in severely stressed soils than in moderately stressed ones (Figure 4b). The severe water deficit suppressed the growth in length of A. senegal shoots but there were only minor differences in shoot fresh weights between the two treatments (Table 1). Thirty days after inoculation all seedlings exposed to severe drought stress were wilted (Figure 5). The severe water deficit retarded or stopped normal nodule development (Table 1, Figure 6). When the numbers of true nodules and their initials were compared between moderately stressed (n = 148) and severely stressed plants (n = 113), the numbers of nodule initials were significantly higher in severely stressed plants (mean = 6.0, SE = 0.69) than in moderately stressed ones (mean = 3.1, SE = 0.33; P < 0.05, non-parametric Mann-Whitney test). The situation was reversed in the case of true nodules (moderately stressed plants: mean = 9.1, SE = 0.75; severely stressed plants: mean = 3.9, SE = 0.52).
Responses of drought-stressed seedlings to the compatible solutes The presence of 0.0003M glycine betaine or trehalose appeared to slightly slow down drying of A. senegal soil mix exposed to severe drought stress (Figure 4a). Compatible solutes maintained also the numbers of culturable rhizobia at the same level as in moderately stressed soil mix (Figure 4b). In the presence of glycine betaine CFU counts of severely stressed soils even exceeded those detected in moderately stressed ones having no increments (Figure 4b). The most important observation was that the presence of 0.0003M glycine betaine in severely droughtstressed soil mix helped A. senegal seedlings to survive. Thirty days after inoculation severely stressed plants grown without any increments were wilted. Instead, when grown with glycine betaine, 70 and 60% of the seedlings were still alive 30 and 42 days after inoculation, respectively. 0.0003M trehalose also tended to keep seedlings alive (Figure 6). Application of compatible solutes seemed to increase shoot growth only under moderate drought stress (Table 1).
Infection process under drought stress Moderate drought stress. The infection process under moderate stress (this work) resembled the one that occurred in optimal growth conditions (Räsänen et al., 2001). A characteristic feature of A. senegal roots grown in moderately stressed jars was that the root hairs did not cover the root and there were many bare roots. After inoculation, not all hairs were deformed but markedly curled hairs also occurred in patches. Deformed hairs were typically dwarfed (15–20 µm), swollen and bent against the root surface (Figure 7a). After incubation of the root systems in the GUS substrate buffer, rhizobia having glucuronidase activity and colonising root surfaces and occupying nodules and infection threads could be clearly distinguished by their dark blue colour (Figure 7b).
Severe drought stress. Because drying of soils occurred gradually and the presence of root hairs and the level of deformation varied a lot between plants even under favourable growth conditions, it was slightly difficult to distinguish exact effects of the water de- ficit on the infection process. However, it appeared that A. senegal seedlings grown under severe drought stress developed less lateral roots but more root hairs than moderately stressed ones. This phenomenon was detected both on inoculated and uninoculated roots. At the beginning of the severe drought stress experiment, the hairs of uninoculated plants usually were long (50– 70 µm) and straight but later the hairs tended to be shorter (<50 µm) and more equal in size, located in bunches. The hairs of the inoculated, severely droughtstressed roots were mostly undeformed (Figure 7c). If the hairs were deformed, part of those were abnormal, being short, swollen but not clearly bent against the root surface (Figure 7d). Nonetheless, properly deformed hairs also occurred in patches. Rhizobia colonising root surfaces could be detected but infected hairs occurred rarely. If there were infection threads, they showed a weak glucuronidase activity (Figure 7e). Nodules were often yellow or brown and showed shrinkage and/or irregularity in shape, suggesting premature senescence. After incubation with the GUS substrate, dark blue colour was irregularly distributed in the nodule (Figure 7f), or detected only after crushing the nodule on the paper, on which the nodule contents produced blue dots. The weak dyeing of infection threads and nodules indicated that either the numbers of rhizobia inside them were reduced or/and rhizobia had a weak glucuronidase activity. In severely stressed soil mix, the presence of 0.0003M trehalose tended to decrease the numbers of root hairs close to the numbers detected on moderately stressed roots. In general, application of compatible solutes into drought-stressed soil mix seemed to increase areas of the roots colonised by rhizobia and the numbers of infected hairs (data not shown) but did not to lead to improved nodulation (Table 1).
DISCUSSION Arid and semiarid soils often suffer from elevated salinity due to the high evaporation. Thus, it was not surprising that many rhizobia isolated from Acacia and Prosopis trees, such as S. arboris strain 1552 used in this study, turned out to be moderately salt tolerant, capable of growing in 0.3–0.5 M (2–3%) NaCl (Craig et al., 1991; Zhang et al., 1991; Zahran et al., 1994, this study). In the case of the salt sensitive S. saheli strain 1496, 0.1 M NaCl stimulated and 0.5 M NaCl and inhibited its growth. PEG 6000 (9, 17 and 24%), which was used to simulate harmful effects of the drought stress (osmotic and matric stresses), reduced growth of sinorhizobia, the more the higher the PEG content was.
Compatible solutes and stressed cultures Depending on the osmotic conditions, rhizobia treat exogenous glycine betaine differently. When grown under low osmotic stress, they use glycine betaine as a carbon and/or nitrogen source, by degrading it progressively to glycine (Bernard et al., 1986). At high osmolarity, the degradation pathway is blocked and glycine betaine is accumulated into the cytoplasm, serving there as a compatible solute (Sauvage et al., 1983; Bernard et al., 1986; Smith et al., 1988; Boncompagni et al., 1999). Except slow-growing B. japonicum, which is considered as an osmosensitive species, the potential to transport glycine betaine and its precursor, choline, is widespread in the family Rhizobiaceae. However, glycine betaine does not function as osmoprotectant for all rhizobia (Boncompagni et al., 1999). In non-stressed conditions, our tropical sinorhizobia could use glycine betaine (0.01) and trehalose (0,01, 0.05 and 0.09M) as a sole carbon source. However, the growth with glycine betaine was delayed compared to that with trehalose. Obviously, not belonging to the sugars, glycine betaine is used and metabolised differently. Under osmotic stress (9 and 17% PEG 6000), all concentrations of glycine betaine and trehalose tested functioned as osmoprotectants for sinorhizobia. Instead, under salt stress only trehalose had a favourable effect. What could explain the phenomenon that Sinorhizobium cultures exposed to osmotic stress produced better growth response with trehalose than with glycine betaine? During the osmotic stress glycine betaine accumulates in cells and it serves as a carbon/nitrogen source only after the relief of the stress (Bernard et al., 1986; Smith et al., 1988). Instead, trehalose is not accumulated in the cytosol but after degradation during the early exponential phase, presumably into two glucose units (Galinski, 1995), it indirectly contributes to raise the levels of two endogenous osmolytes, glutamate and NAGGN (Gouffi et al., 1999). Thus, capability of trehalose to serve both as a carbon source and osmoprotectant explains the better growth improvement of trehalose. Moreover, trehalose might have favoured EPS production, and thus, increased the optical density values.
Compatible solutes and drought stress Sinorhizobial cells. When the GUS marked strain 2180, a derivative of S. arboris strain 1552, was grown in the severely drought-stressed A. senegal soil mix, the numbers of culturable rhizobia were significantly reduced from 107 to 106 CFU g−1, approximately. However, the application of 0.0003 M glycine betaine and trehalose in the A. senegal soils maintained the cell numbers at the same level as those detected in moderately stressed soils. The advantageous role of glycine betaine on the cell numbers might have varied during the course of our plant experiment. In moderately drought-stressed soil mix and at the beginning of the severe drought experiment rhizobia probably used glycine betaine as a carbon and/or nitrogen source. Later, when rhizobia started to suffer from severe water deficit, degradation of glycine betaine was blocked and glycine betaine was used as a compatible solute (Sauvage et al., 1983; Bernard et al., 1986; Smith et al., 1988; Boncompagni et al., 1999). The favourable effect of trehalose on rhizobial numbers was probably based on it serving as a carbon source or protecting them from desiccation, by stabilising membranes and whole cells (Bushby and Marshall, 1977; Potts, 1994).
A. senegal seedlings. A. senegal is an extremely drought tolerant tree species, succeeding in areas with rainfall as low as 100 mm year−1. Adult A. senegal trees develop deep tap roots, through which they can reach moist soil layers or the ground water located 10–30 m from the soil surface (Hocking, 1993). Young Acacia seedlings can also to some extent adapt to water deficiency, for example, by increasing their root:shoot ratio, resulting in enlargement of the absorptive root biomass (Michelsen and Rosendahl, 1990; Otieno et al., 2001). In our work, A. senegal seedlings grown under severe drought stress appeared to develop more root hairs than moderately stressed or non-stressed ones (Räsänen et al., 2001), obviously, in order to intensify water uptake in their immediate proximity. A. senegal seedlings. Regarding glycine betaine, Rhodes and Hanson (1993) have grouped plants into non-accumulators (<1 µmol g−1 dry wt) and accumulators (under non-stressed conditions 5–100 µmol g−1 dry wt and under natural or experimental saline or dry conditions 40–400 µmol g−1 dry wt). Acacias can be considered as non-accumulators, because nonstressed acacias have contained glycine betaine less than 1 µmol g−1 dry wt (Rhodes and Hanson, 1993; this study), and glycine betaine content in droughtstressed Australian acacias was 8.5 µmol g−1 dry wt (Erskine et al., 1996). Our work indicated that A. senegal seedlings were capable of translocating foliar-applied glycine betaine into the root system. Thus, it was possible to investigate if exogenously applied glycine betaine would improve drought tolerance of A. senegal seedlings. The presence of 0.0003 M glycine betaine in the soil mix helped A. senegal seedlings to survive under severe drought stress. Previous studies indicated that the favourable effect of exogenously applied glycine betaine on crop plants is based on their relatively lower daily transpiration rates and on the maintenance a better water status (Agboma et al., 1997; Xing and Rajashekar, 1999). In our plant experiments, treated soils tended to have more water left compared to untreated ones. This phenomenon together with previous data suggests that with the help of glycine betaine the drought-stressed A. senegal seedlings were able to control water uptake and transpiration more efficiently. It is supposed that bacterial EPS protects bacteria from desiccation by altering their microenvironment (Roberson and Firestone, 1992). Bacterial EPS may also absorb water (Potts, 1994). Inoculation of plant roots with an EPS-producing Rhizobium strain was reported to improve soil structure (Alami et al., 2000). Altogether, an additional explanation for the improved survival of the drought-stressed A. senegal seedlings with compatible solutes could be that the increased rhizobial population having enhanced EPS production and/or other metabolic activities colonises roots and rhizosphere, subsequently protecting roots from the drought stress.
A. senegal-Sinorhizobium symbiosis under drought stress Similarly to heat stress (Räsänen and Lindström, 1999), the root hairs of severely drought-stressed A. senegal seedlings were deformed. There were only few infection threads and the development of nodule initials into proper nodules was prevented, showing consistency with observations with herbaceous legumes exposed to drought stress (Worral and Roughley, 1976; Gallacher and Sprent, 1978). In our work, application of compatible solutes did not assist development of symbiosis. However, like under heat stress (Räsänen and Lindström, 1999), A. senegal roots exposed to severe drought stress had the most important elements that were needed for the proper infection, i.e., lateral roots, root hairs, sufficient numbers of effective rhizobia (105–107 cfu g−1 of soil) and deformed root hairs (indicating production of rhizobial Nod factors). Similar observations were done in drought-affected soybean roots (Williams and Sicardi de Mallorca, 1984). It appears that formation of nodules is a rare event even under non-stressed conditions. Infection threads develop only on a very small proportion of the hairs and subsequently, less than half of the infected hairs result in nodules (Dart, 1977; Wood and Newcomb, 1989). Obviously, environmental stresses decrease the potential for nodulation drastically. Which factor(s) could then explain the inhibition of nodulation? Generally it is the plant being a more sensitive partner that restricts nodulation. Perhaps the plant hormone abscisic acid (ABA), the production of which is associated with drought and saline stresses (Bray, 1997), may contribute to the extensive inhibition of nodulation (Williams and Sicardi de Mallorca, 1984).
CONCLUSIONS Mechanisms connected to drought and osmotolerance in plants and bacteria are complex. Our work showed that by application of glycine betaine or trehalose it is possible to improve drought tolerance of tropical sinorhizobia (both) and that of A. senegal seedlings (glycine betaine). However, the fact whether compatible solutes protect plants or bacteria from drought also in the field, needs more studies.
ACKNOWLEDGEMENTS We thank Elena Lapina-Balk for skilful assistance, Katriina Pullianen, Danisco Sugar Oy, Development Center, Finland, for HPLC analyses of glycine betaine, Sudan Seed Center for providing A. senegal seeds, Kirsti Tiihonen for inspiring discussions and Ken Giller for his useful comments Academy of Finland, Kemira Foundation and University of Helsinki supported this work.
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