Ptant, Cetl and Environment {^995) 18, 801-806 Enhancement of seed germination in high salinity by engineering mannitol expression in Arabidopsis ttiaiiana J. C. THOMAS,'-^* M. SEPAHI,' B. ARENDALL' & H. J. BOHNERT''" ' Department of Biochemistry, and Departments of ^ Plant Sciences, and ^Molecular and Cellular Biology, The University of Arizona, Biosciences West, Tucson, AZ 85721, USA ABSTRACT The bacterial gene mtlD, which encodes mannitol 1-phosphate dehydrogenase (E.C.I.1.1.17), was transformed into Arabidopsis thaliana and expressed under control of the CaMV 35S promoter. AfrfD-transformants accumulated mannitol, a sugar alcohol that is not normally found in Arabidopsis. Amounts of soluble carbohydrates, sucrose, glucose, fructose, myo-inositol and mannitol were determined in different tissues of wild-type and transgenic plants. We estimated that less than 1% of the carbon assimilated was converted into mannitol by the transgenic plants. The establishment of individual transformed lines (after self-crossing three times) resulted in high and low mannitol-producing lines which were stably maintained. The presence of mannitol did not alter plant appearance or growth habit. When MtlD-expressing seeds and control seeds (T3 generation) were imbihed with solutions containing NaCI (range 0 to 400 mol m~'^), transgenic seeds containing mannitol germinated in medium supplemented with up to 400 mol m"'' NaCI, while control seeds ceased germination at 100 mol m"^ NaCI. It is doubtful whether the ability to germinate in high salt was a result of an osmotic effect exerted by elevated levels of mannitol, considering that mannitol concentrations were in tbe mol m~^ range in seeds. A specific effect of polyols, for example on the integrity of subcellular membranes or enzymes, cannot be excluded. Key-words; metabolic engineering; polyol; sugar alcohol. Abbreviations; CaMV35S, promoter for Cauliflower mosaic virus 35S RNA; K,,,"*. kanamycin resistance; nitlD, gene encoding Mtl-DH frotn Escherichia coli; Mtl-DH, matinitol 1-phosphate dehydtogenase (E.C. 1.1.1.17); uidA, gene encoding /3-glucuronidase from E. coli. INTRODUCTION The accumulation of polyols in response to stresses that affect water availability and the accumulation of other *Correspondence and present address; John C. Thomas, Department of Natural Sciences, University of Michigan (Dearborn), Dearborn, MI 48128-1491, USA. © 1995 Blackwell Science Ltd metabolites, such as proline and glycine-betaine, have been described in many studies (Flowers et al. 1977; Ford 1984; Thomas et al. 1992; Vernon & Bohnert 1992; Delauney & Verma 1993; Hanson etal. 1994). Accumulation of these compounds is strongly correlated with resistance or tolerance of plants to abiotic stresses that affect water availability. Proposed mechanisms of action of these accumulating substances include their action in osmotic adjustment, and/or their action as compatible solutes or osmoprotectants (McCue & Hanson 1990). In osmotic adjustment during NaCI stress, for example, an accumulating metabolite on a mole-by-mole basis would replace other compounds within the cell. Sodium or other toxic compounds might be sequestered into the vacuole, if the metabolite were strictly confined to the cytoplasm. If found in the cytosol, organelles and vacuole, the metabolite would increase the osmotic pressure of the cell. For example, when mistletoe taps into the host phloem, polyol accumulation provides the parasite with a high osmotic pressure (Richter & Popp 1992). Thus, when acting as compatible solutes during osmotic adjustment, accumulating metabolites are considered non-inhibitory to cellular metabolism and their effect might be exerted at high or moderately high concentrations. Osmoprotectants could even act at low concentrations by protecting specific structures or enzymatic processes, by exerting regulatory effects on ion or water uptake or transport, or by stabilizing multi-subunit enzyme complexes or membranes (Smirnoff & Cumbes 1989; Sommer etal. 1990- Smirnoff 1993). ,o,;j-. rrjn. We have begun to analyse the effects of one class of putatively protective metabolite, polyols, on the performance of plants under stress (Tarczynski et al. 1992; Tarczynski et al. 1993; Vernon et al. 1993). A bacterial gene, mtlD, encoding mannitol 1-phosphate dehydrogenase, which catalyses the production of the acyclic polyol mannitol, was transfeiTed to tobacco. We showed that the transgenic plants accumulated mannitol. These plants under controlled growth conditions were able to tolerate moderate salt stress better than control plants. The function(s) of the polyol, however, and the mechanism(s) through which mannitol might act are still unknown. Instead of monitoring whole-plant performance under stress, a different investigation was performed here. By 801 802 J. C. Thomas et al. engineering mtlD into Arabidopsis thaliana and selfmg selected transformed lines, large populations of plants were grown. Mannitol production by itself did not alter the appearance and growth of these plants relative to control plants. We analysed the distribution of sugars and polyols in different tissues and in seeds of MtlD and control Arabidopsis. The results indicated that high NaCI was less stressful at germination even at relatively low concentrations of mannitol in the germinating MtlD seeds and seedling when compared to controls. All lines germinated equally well on 100 mol m""^ NaCI, in contrast to wildtype. Different lines, however, showed differences in the maximal concentration of NaCI tolerated. Since the amount of accumulated mannitol in seeds was lower than that required to exert an osmotic effect, we suggest that this polyol might act as an osmoprotectant rather than as a compatible solute in Arabidopsis thaliana during germination under non-physiological salt conditions. , >ififo fitd tions, up to 500 seeds were sown onto petri dishes and getmination was scored after 7 d of germination under conditions as described above. Seeds were scored as germinated when the radicle was at least 5 mm long and the cotyledons had begun to unfold. Seeds were surface-sterilized and germinated in previously sterilized 1:1 vermiculite/ potting soil. Individual plants were grown in 5 cm containers (24 °C; 24 h light at 150-250 jimoX nf^ s"' photon flux density). Different plant lines were grown in a growth chamber and roots, leaves, stalks, flowers and siliques were harvested at the same time for sugar, proline and polyol analysis. HPLC analysis The analysis of sugars and sugar alcohols was performed accordingto Adams era/. (1992, 1993, 1995). MATERIALS AND METHODS RESULTS Gene constructs, transformation, and regeneration of plants The gene construct used consisted of a CaMV 35S promoter transcriptionally fused to a short segment upstream of the start codon of the bacterial mtlD gene as described (Tarczynski et al. 1992). After root transformation of Arabidopsis thaliana (Landsberg erecta) a number of individual transformants were selected on 50 /ig cm"-* kanamycin (Valvekens et al. 1988). The first generation was allowed to .set seed in tissue culture and Tl seeds were recovered. From these seeds, plants were grown to maturity and selfing was repeated. Progeny were selected on 100 /ig cm"-^ kanamycin and populations of plants and T3 seeds were established. From such T3 seeds, plants were grown and analysed for the presence of mannitol and sugars in different organs. The bacterial gene mtlD (Davis et al. 1988) was engineered to contain a CaMV 35S promoter/enhancer element and the 3' end of the NOS (nopaline synthase) gene as described (Tarczynski (?r a/. 1992). Arabidopsis thaliatia (ecotype Landsberg erecta) were transformed using root explants (Valvekens et al. 1988). Young roots were incubated with Agrobacterium tumefaciens containing the 35S-mtlD gene for 30 min. After washing and co-cultivation for 2-3 d, the roots were transferred to liquid medium containing 500 /lg cm~" vancomycin and 50 fig cm '^ kanamycin and washed several times. The roots were then placed on solid medium with vancomycin and kanamycin (24-27 °C, 16 h light at 150-200 /imol m"^ s'' photon flux density) for 2-3 weeks. Green shoots which emerged were transferred to growth medium (200 /ug cm "* vancomycin, 50 /lg cm'"* kanamycin). Plants were allowed to set seeds in sterile culture. Viable seeds were recovered from mote than 20 individual transformation events and seeds were germinated on 100 /lg cm^"' kanamycin. Subsequent green K,,,'* progeny were grown to maturity and selfed. This step was repeated once to obtain the T3 generation seeds. As a control, the gene encoding ^-glucuronidase (uidA) (Jefferson et al. 1987) under 35S promoter control was transferred into Arabidopsis using the same procedure. Growth parameters and germination studies Surface-sterilized seeds were placed individually on agar containing MS-salts, 1% sucrose, 0-1 mg dm""^ thiamine HCl and 0 8% Bacto agar. NaCI was added at different concentrations (0 to 400 mol m"^). Approximately 100 seeds were placed on each 8 cm petri dish and incubated in a growth room (24-27 °C, 16h light at 150-200 /imol m~^ s photon flux density). To obtain data on larger popula- Extracts from wrrfA-transformed control and /«r/D-transfonned T3 plants were analysed by HPLC at high pH with pulsed amperometric detection of hydtoxyl gtoups (Adams et al. 1992, 1993, 1995). The uidA-expressing (GUS expression; Jefferson et al. 1987) plants showed sugar profiles identical to those of wild-type plants (Fig. la). Extracts frotn mr/D-transformed plants showed a typical distt ibution of sugars and contained one additional prominent peak, with a retention time -2-2 min, not found in non-transformed controls (Fig. lb). NMR analysis of collected material from this peak (data not shown) indicated that the material in this peak was mannitol. The amount of mannitol in individual lines was variable, similar to findings with transgenic tobacco (Tarczynski et al. 1992; 1993). Levels ranged ftom 0-05 to 12 00 /imol g~' fresh weight (Table 1). This amount did not seern to be correlated with the amounts of the other sugars. Table 2 shows this distribution of sugars and mannitol in different tissues of transformed plants from a representative experiment. Wild-type values for sugars are comparable, varying from 1 to 17 /itnol g ' fresh weight for sucrose. In leaves of transgenic mtlD plants, the amount of sucrose ranged from 0 4 to 1 5 /imol g"' fresh weight, that of glucose from © 1995 Blackwell Science Ltd, Plant, Cell and Environment, 18,801-80 Engineering of mannitol expression in Arabidopsis nA 200 nA 200 M 100 9.6 I 0 6 Mm 6 Figure 1. HPLC profiles of soluble low-tnolecular-weight carbohydrates in wild-type and transgenic Arabidopsis thatiana expressing the bacterial mannitol I-phosphate dehydrogenase gene. The analysis of sugars, proline and polyols was performed according to Adams et al. (1992). I, inositol; G, glucose; F. fructose; S, sucrose; M. niannitol; P. proline (detectable levels only under stress, panel c). (a) Control plants containing 35S-«iVM gene, (b) Plants containing the ?i5S-nittD Gene, (c) Plants containing the mtlD gene after 5 d in 200 mol m""" NaCI. 0-6 to 2-7 /imol g ' fresh weight, and that of fructose from 0 2 to 13 /lmol g~' fresh weight. All samples were taken during the second half of the light period. The analysis of a large number of control or mannitol-containing plants showed that notmal sugar and tnannitol amounts were within a range of approximately 0 to 10 /imol g~' fresh weight with large variations depending on plant age and tissue sampled. There was no significant difference between the lines. Under stress conditions, plants growing in the presence of 200 mol m""* NaCI showed changes in sugars (Fig. lc). The amounts of sucrose and reducing sugars declined drastically, depending on the severity of the stress and also on the time during development at which the stress was © 1995 Blackwell Science Ltd, f/fld/, Cell and Environment, 18.801-806 803 applied. As a result of the decline in sugar content, the relative contribution of mannitol to the osmotic pressure increased and several other, minor carbohydrate peaks, whose nature we have not studied, increased. In addition, proline was easily detected under stress conditions (Fig. lc). The identification of the material in this peak (retention time 3-45 min) as proline is based on the retention time in comparison with proline standards, re-chromatography of the material on a cation HPLC system (W. Jones, The University of Arizona, personal communication), and on the fact that the material could be removed by desalting of the extracts (data not shown). To test for the ability to germinate under stress conditions, control and mannitol-expressing seeds were surfacesterilized and placed on agar containing levels of NaCI ranging from 0 to 400 mol m"-' NaCI (Fig. 2). The control seeds, which had been transformed with a 35S-uidA gene, generally ceased germination at levels higher than 100 mol m"-', similar to teports for wM-type Arabidopsis (Saleki et al. 1993). Lines which contained mannitol always germinated better than controls on 100 mol m~-^ NaCI. At 200 mol m"'' NaCI some lines were comparable to wildtype germination on 100 mol m""" NaCI, and a few lines did germinate, albeit poorly, on 400 mol m^"* NaCI (Fig. 2). Figure 3 provides an impression of the germination and growth of 35S-M(V/A-expressing and 35S-tntlD expressing Arabidopsis thaliana populations in the absence and presence of NaCI. Non-transfotmed wild-type plants behaved identically to the 35S-uidA plants (data not shown). Germination on agar supplemented with MS salts and that on agar supplemented with I % sucrose were identical, apart from differences in the overall germination of the lines, in the absence of NaCI (Fig. 3, top row). In the presence of 200 mol m"^ NaCI, 35S-M(<a'A plants did not germinate at Germination 0 mol m"^ NaCI Germination 100 mol m"^ NaCI Germination 200 mol m'^ NaCI Germination 400 mol m"-^ NaCI 0.0% 121.la M3 M5 Plant Line M6 Figure 2. Germination of wild-type and Iransgenic Arabidopsis Ihaliana in the absence or presence of NaCI. Percentages given are with tespect to the total number of seeds. The experiment was performed two times with different seed batches. Seeds from line M2 only showed some germination on 400 inol m ' NaCI. 804 J. C. Thomas et al. Table 1. Mannitol in Arabidopsis thaliana expressing 35S-mtlD and 'i5S-uidA Plant clone Lower leaves Upper leaves Ml 0-05 (0-08) 2-50 (0-21) 0-17 (0-05) 1-75 (0-21) 0-24 (0-12) 5-80 (1-60) 3 07(1-10) 4-05 (0-63) 0 32 (0-08) 1-59 (014) 0-66 (014) 0-43 (0-07) 0-28 (0-14) 5-63 (0 92) 3-14(1-04) 2-99 (0-29) ND ND ND ND ND ND ND ND M2 M3 M4 M5 M6 M7 M8 M9 12M-a 12M-b 12M-C Stem Flower Silique ND ND ND ND ND 0-10(0-04) 0-72(0-10) 0-56 (0-06) ND ND 3-0(0-70) 2-68 (0-40) ND ND ND ND ND 0-21 (0-03) ND 7-47(1-10) 5-90 (0-50) NT ND ND ND ND ND 7-37(1-10) 12-00(2-20) NT ND ND ND ND NT 0-84(0-15) 7-20(0-55) 1-70(0-61) 0-21 (0-10) 0-70 (0-09) ND Germinated on 200 trol tir^^ NaCI Seeds ND 10-05(0-97) 9-95(1-90) 4-25 (0-35) ND ND ND ND 0-51 (0-05) 0-36 (0-05) NT NT NT NT NT ND ND NT NT Extracts of soluble carbohydrates were made frotn T3 pooled plants of independent K^," sample lines. Lines 121-1 are lines from independent transformation events with a 35S-uidA construct (T3 after Km" selection). Data are expressed as /(mol mannitol g~' fresh weight (or dry weight for seeds). NT: not tested; ND: none detected. Standard ertors are given in parentheses. In this experiment NaCI was used at 200 mol m"^ in agar with MS salts. The experiment was performed three times with different seed batches. ' ! • . ' • / • all (Fig. 3, bottom row, right), while 35S-mtiD line M2 (Table 1) germinated at approximately 40% (Fig. 3, bottom, middle), and line M3 (Table 1) germinated poorly (Fig. 3, bottom, left) at approximately 14%. Transgenic plants germinating in 200 mol m "* NaCI could be rescued, if they were removed from the plates within approximately 2 weeks. However, rescue was no longer possible once plants had developed more than approximately four true leaves on medium containing 200 mol m"'' or higher NaCI. DISCUSSION Figure 4 outlines the pathway which results in the accumulation of mannitol in a plant that does not normally synthesize this polyol due to mtlD expression. In E. coli (Fig. 4, inset), from which the mtlD gene is derived, the pathway proceeds through the uptake-coupled phosphorylation of mannitol to mannitol 1-phosphate (M-IP). The MTL-D enzyme converts M-IP to fructose 6-phosphate (F-6P) which becomes available for catabolic reactions. In transgenic mtlD plants, the enzyme converts part of the F-6P to M-1P utilizing cytosolic NADH + H"^. M-1P is dephosphorylated, probably through the action of a broad-substrate phosphatase which we have not characterized. Indeed, elevated levels of M-IP or reduced levels of F-6P have not been detected in transgenic tobacco (R.G. Jensen, The University of Arizona, personal communication). Once produced, we think that mannitol is confined to the cells in which it is made or that it leaves cells slowly. No information has been obtained on whether mannitol may be transported out of the cells. An alternative is that mannitol may be re-phosphorylated and may again enter the pathway to sucrose. Experiments which address this question are underway. We surmise that mannitol is slowly catabolized because accumulation reaches a plateau, and the plateau value is not significantly increased when enzyme amounts are increased by crosses of different lines (data not shown). The localization of mannitol in cells is not known. If it were confined to the cytosol, a concentration of mannitol of approximately 100 to 150 mol m"^ would result in some lines.^'However, if mannitol were to move into the vacuole, concentrations in total cell water might be as low as 10 mol m"''. Considering that mannitol accumulation and the ability of a transgenic seed to germinate in elevated NaCI levels were coincident, how might mannitol act during germination at high salinity? Osmotic adjustment as the mechanism of mannitol action is conceivable only when mannitol is restricted to the cytoplasm. Even under these circumstances, the amount of mannitol, equivalent to approximately 100 mol m"'', is barely high enough to fulfil the requirement of an osmolyte for osmotic adjustment. Table 2. Representative determinations of principal sugars in leaves of control (pBI 121 -1) and MtlD (M)-transformed Arabidopsis Sample Mannitol Glucose Fructose Sucrose 121-1-a 12M-b ND ND 0-6 1-5 1-3 1-7 0-9 0-6 2-7 1-0 3-9 1-7 2-7 1-4 0-7 0-7 0-8 I-O 1-7 1-0 0-7 0-7 0-5 Ml M2 M3 M4 M5 M6 M7 1-7 0-8 1-3 0-2 0-6 0-3 0-8 0-6 0-4 0-8 1-5 1-0 0-4 Data are from a single, representative experiment, and are presented in /lmol g"' fw for each sugar/polyol. ND: not detected. © 1995 Blackwell Science Ltd, Plant, Cell and Environment, 18, 801-80 Engineering of mannitol expression in Arabidopsis Figure 3. Photograph of petri dishes showing Arabidopsis seedlings germinating in the absence (top row) or presence (bottom row) of 200 tnol m""* NaCI. The dishes on the right (top/ bottotn) are 35S-uidA. those in the middle (top/ bottom) are line M2, and those on the left (top/bottom) are line M3. although mannitol could act in concert with the proline that is accumulating as part of the normal water and salinity stress response of Arabidopsis. Solutes that accutnulate in response to an osmotic challenge range from inorganic and organic cations and anions to specific amino acids, sugars, sugar alcohols and tertiary ammonium or sulphonium compounds (Yancey et al. 1982; Smirnoff 1993; Hanson et al. 1994). Solute accumulation is found in bacteria, fungi, plants and animals, indicating that most or all organisms are capable of osmotic adjustment to sotne degree. Possibly, the marginal increase of mannitol achieved with the present gene constructions elevates osmolyte concentration beyond a threshhold value that confers increased tolerance. In a germinating seed, the concentration of mannitol detected was higher than in turgescent mesophyll cells. It may be that this increase in osmolarity provides an advantage during germination, either by facilitating water uptake or by preventing, in the short term, excessive sodium influx. In both scenarios the tnovement of solutes across membranes is involved. Mannitol and other osmoprotective substances may influence the transport of metabolites and solutes across metnbranes in a specific way. There have been several suggestions about possible functions of osmoprotectants: radical scavenging, protection of enzymes, enzyme complexes or membranes, and a sink for ATP NADPH I 4 pholotynthatfs chloroplast Figure 4. Engineered pathway dependent on the action of mannitol I -phosphate dehydi ogenase in transgenic Arabidopsis thaliana. For explanations, see the Discussion. © 1995 Blackwell Science Ltd. Plant, Cell and Environmem, 18. 801-806 805 806 J. C. Thomas et al. To ' photosynthetically assimilated carbon under stress (Lewis & Smith 1967; Briens & Larher 1983; Smirnoff & Cumbes 1989; Krall etal. 1989). Production of mannitol could promote seed germination in several ways, for example by conferring resistance to NaCI toxicity and/or by allowing seeds to absorb greater levels of water. Clearly, more work is needed to investigate the mechanisms by which osmoprotectants act. ACKNOWLEDGMENTS We thank Pat Adams and Wendy Jones for HPLC analyses, and Richard G. Jensen and Pat Adams for suggestions on the manuscript. This work was supported by grants from DOE (Biological Energy Research) and U.S. Department of Agriculture (Plant Responses to the Environment) to H.J.B. and from Arizona Agricultural Experiment Station to H.J.B. and J.C.T. REFERENCES Adams P., Thomas J.C, Vernon D.M.. Bohnert H.J. & Jensen R.G. (1992) Distinct cellular and organismic responses to salt stress. Plant and Celt Physiotogy 33, 1215-1223. 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Science 217, 1214-1222. Received 9 September 1994; received in revisedform 21 December 1994; accepted for pubtication 18 January 1995 © 1995 Blackwell Science Ltd, Plant, Cell and En vironment, 18, 801 -806