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Glycinebetaine: an effective protectant against abiotic stress in plants
Review
Glycinebetaine: an effective protectant against abiotic stress in plants Tony H.H. Chen1 and Norio Murata2 1 2
Department of Horticulture, ALS 4017, Oregon State University, Corvallis, OR 97331, USA National Institute for Basic Biology, Okazaki 444-8585, Japan
Glycinebetaine (GB) has been studied extensively as a compatible solute because of the availability of GBaccumulating transgenic plants that harbor a variety of transgenes for GB-biosynthetic enzymes. Both the exogenous application of GB and the genetically engineered biosynthesis of GB increase the tolerance of plants to abiotic stress. As reviewed here, studies of such increased tolerance to abiotic stress have led to considerable progress in the characterization of the roles of GB in stress tolerance in plants. In particular, the reproductive organs of GB-accumulating transgenic plants exhibit enhanced tolerance to abiotic stress. Furthermore, accumulation of GB results in increased yield potentials under non-stress conditions. Glycinebetaine (GB), an effective protectant against abiotic stress GB is a fully N-methyl-substituted derivative of glycine, and it is found in a large variety of microorganisms, higher plants and animals [1]. GB belongs to a group of compounds that are known collectively as ‘compatible solutes’: small organic metabolites that are very soluble in water and non-toxic at high concentrations. GB accumulates in the chloroplasts and plastids of many halotolerant plants. These plants accumulate high levels of GB in response to abiotic stress [2,3]. Levels of accumulated GB are generally correlated with the extent of stress tolerance [1]. GB effectively stabilizes the quaternary structures of enzymes and complex proteins, and it maintains the highly ordered state of membranes at non-physiological temperatures and salt concentrations [4]. Both the exogenous application of GB (Table 1) and the introduction, via transgenes, of the GB-biosynthetic pathway into plants that do not naturally accumulate GB (Table 2) increase the tolerance of such plants to various types of abiotic stress. This increased tolerance to abiotic stress provides useful systems for investigating the mechanisms by which GB protects plants against abiotic stress. Among the compatible solutes, GB is a particularly effective protectant against abiotic stress [5–7]. Studies of GB have focused on GB-mediated tolerance to various kinds of stress and at various stages of the life cycle of plants [8,9]. Exogenous application of GB Exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stress, and it can enhance subsequent growth and yield (Table 1). Corresponding author: Murata, N. ([email protected]).
When applied to leaves of plants, GB is readily taken up by leaf tissues [10]. GB can also be taken up via roots [11]. When GB is applied to the leaves of tomato (Lycopersicon esculentum) plants, most of the GB that is taken up by the leaves is localized in the cytosol and only a small fraction of the cytosolic GB is translocated to chloroplasts [10]. When [14C]glycinebetaine was applied to leaves of summer turnip (Brassica rapa L.) plants, the radio-labeled GB was translocated to roots within two hours [11]. During the next 24 h, the radio-labeled GB was translocated to all parts of the plant. In tomato plants, large amounts of foliar-applied GB were translocated to meristem-containing tissues, including the flower buds and shoot apices. It was demonstrated that GB was translocated to actively growing and expanding portions of plants, the long-distance translocation of GB being mediated by the phloem [12]. Reactive oxygen species (ROS) are produced continuously as products of various metabolic pathways, even when plants are growing under non-stress conditions. These ROS are scavenged by a variety of antioxidant defense systems that prevent ROS from reaching toxic levels. All forms of abiotic stress, including high salt, chilling, freezing and drought stress, cause an oxidative burst in plant cells. The application of hydroxyl radicals (OH*) to Arabidopsis roots resulted in a massive, dosedependent efflux of K+ ions from epidermal cells in the elongation zone [13]. However, the presence of GB at 5 mM in the incubation medium significantly reduced this efflux of K+ ions. Furthermore, in tomato plants, exogenously applied GB significantly reduced the chilling-induced production of H2O2 [10]. Because GB does not scavenge ROS directly, GB must mitigate the damaging effects of oxidative stress in other ways, for example by activating or stabilizing ROS-scavenging enzymes and/or repressing the production of ROS by an unknown mechanism. Genetically engineered biosynthesis of GB GB is synthesized either by the oxidation (or dehydrogenation) of choline or by the N-methylation of glycine [14]. Genes that encode GB-biosynthetic enzymes have been cloned from various microorganisms and plants, and transgenic plants of various species have been produced that express one or several of these genes (Table 2). The transgenic plants accumulate GB at a variety of levels and exhibit enhanced tolerance to a variety of abiotic stresses (Table 2). In plants that do not accumulate GB naturally, the highest levels of accumulated GB have been found in leaves of codA-transgenic rice (Oryza sativum) plants
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Table 1. Enhancement of tolerance to abiotic stress via exogenously applied glycinebetaine in higher plants Plant species Arabidopsis thaliana
Avena sativa Brassica rapa Fragaria X ananassa Hordeum vulgare Lycopersicon esculentum
Medicago sativa Oryza sativa Phaseolus vulgaris Triticum aestivum Zea mays
Types of stress Salt Freezing Freezing Oxidative Chilling Drought, salt Drought, salt Drought, salt Freezing Drought, salt Chilling Drought Drought, salt Drought, salt Freezing Salt Drought Freezing Drought, salt Chilling
Environment
Refs
Growth chamber Greenhouse Growth chamber Growth chamber Growth chamber Greenhouse, field Greenhouse Greenhouse, field Greenhouse Greenhouse, field Growth chamber Field Growth chamber Greenhouse Growth chamber Growth chamber Greenhouse Growth chamber Greenhouse, field Growth chamber
[44] [45] [46] [13] [40] [12] [47] [12] [48] [12] [5,10,11] [49] [50] [47] [51] [52] [53] [2] [12] [54]
[5.3 mmol g 1 fresh weight (FW)] [15]. In a natural GBaccumulator, maize (Zea mays), the highest reported level of GB in leaves of betA-transgenic maize plants is 5.7 mmol g 1 FW [16,17]. Thus, in terms of maximum levels of GB in transgenic plants, there appears to be no significant difference between plants that accumulate GB naturally and those that do not. Furthermore, glycinemethylation enzymes, such as ApGSMT and ApDMT (Aphanothece halophytica glycine sarcosine methyltrans-
ferase and A. halophytica dimethylglycine methyltransferase, respectively; see Box 1), do not have any apparent advantages over choline-oxidizing enzymes in efforts to engineer the biosynthesis of GB in plants [18]. GB-producing transgenic rice lines were generated in which the cox gene for choline oxidase from Arthrobacter pascens, fused to a chloroplast-targeting sequence, was expressed under the control of either an abscisic acid (ABA)-inducible promoter [a stress-inducible promoter (SIP)] or the promoter of a constitutively expressed gene for ubiquitin (UBI) [19]. The highest level of GB that accumulated under high-NaCl conditions [2.60 mmol g 1 dry weight (DW)] in SIP lines was not as high as that in UBI lines (3.12 mmol g 1 DW). Therefore, the use of an ABA-inducible promoter was not particularly effective for production of GB de novo. Accumulation of GB in chloroplasts Two major factors that influence the tolerance of plants to abiotic stress are the concentration and the localization of GB in the cell. In many studies of the engineered accumulation of GB in plants, GB-biosynthetic enzymes have been targeted to chloroplasts. In a few studies, the enzymes have been targeted to either the cytosol or the mitochondria, or to both the cytosol and the chloroplasts simultaneously (Table 2). In codA-transgenic plants, the gene product has been targeted predominantly to chloroplasts [14]. The resultant transgenic plants accumulate GB primarily in their chloroplasts, and they exhibit tolerance to various kinds of abiotic stress [5–9]. In rice plants transformed with a chloroplast-targeted codA construct, the photosynthetic
Table 2. Transgenic plants engineered to synthesize glycinebetaine and their enhanced tolerance to abiotic stress Plant species Arabidopsis thaliana
Gene codA codA codA codA codA codA cox CMO +BADH ApGSMT +ApDMT cox Brassica napus codA Brassica juncea codA Diospyras kaki betA Gossypium hirsutum Lycopersicon esculentum coda
Nicotiana tabacum
Oryza sativa
Solanum tuberosum Zea mays
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Subcellular location Chloroplasts Chloroplasts Chloroplasts Chloroplasts Chloroplasts Chloroplasts Cytosol Unknown Cytosol
Maximum accumulation 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.9 mmol g 1 DW 900 mmol g 1 DW 2.0 mmol g 1 FW [+5 mM glycine] Cytosol 13 mmol g 1 DW Chloroplasts 0.82 mmol g 1 FW Chloroplasts 0.3 mmol g 1 FW Cytosol 354.3 mmol g 1 DW Chloroplasts 0.3 [leaf] mmol g 1 FW 1.2 [Flower] mmol g 1 FW Chloroplasts 23 nmol mg 1 chlorophyll codA Cytosol 95 nmol mg 1 chlorophyll codA Chloroplasts + cytosol 120 nmol mg 1 chlorophyll codA cox Cytosol 13 mmol g 1 DW Cytosol 35 nmol g 1 FW betA Chloroplasts 4.6 mmol g 1 FW BADH coda Cytosol 5.3 mmol g 1 FW Chloroplasts 1.1 mmol g 1 FW codA 5.1 mmol g 1 FW betA (modified) Mitochondria Chloroplasts 3.1 mmol g 1 DW cox Chloroplasts 0.43 mmol g 1 DW CMO Chloroplasts 2.12 mmol g 1 DW codA codA Chloroplasts 1.43 mmol g 1 FW betA Cytosol 5.7 mmol g 1 FW Cytosol 5.7 mmol g 1 FW betA
Enhanced tolerance Chilling Chilling, salt Heat Strong light Freezing Salt Salt, drought, freezing Salt Salt, chilling CuSO4 Drought, salt Salt Salt Drought Chilling Salt Chilling, salt Oxidative Salt Salt, chilling Heat Salt, chilling Salt, chilling Salt, drought Salt Salt, day/night temperature (28/13 8C) Salt Oxidative, salt, drought Chilling Drought
Refs [21] [24] [22] [55] [46] [8,44] [56] [57] [18] [56] [58] [59] [60] [9] [20]
[56] [61] [31,35] [15] [15] [62] [19] [63] [64] [65] [16] [17]
Review Box 1. Accumulation of glycinebetaine (GB) in the halotolerant cyanobacterium Aphanothece halophytica Cyanobacteria are prokaryotes with photosynthetic machinery that resembles that of plants both structurally and functionally [42], and these cyanobacteria are capable of oxygenic photosynthesis. Cyanobacteria have successfully colonized a wide range of environments. Several strains of cyanobacteria grow naturally in hypersaline water, such as the Dead Sea and the Solar Lake in the Sinai Desert [42]. The most halotolerant species can grow in concentrations of NaCl as high as 3 M [42,43]. These cyanobacteria are extremely tolerant to salt stress and synthesize and accumulate GB in their cytoplasm [42,43]. The sustained growth under salt stress of Aphanothece halophytica, an extremely salt-tolerant cyanobacterium isolated from the Dead Sea, is mediated by the accumulation of GB [42,43]. GB is the major compatible solute in this cyanobacterium [43]. Most known biosynthetic pathways to GB include the twostep oxidation of choline [14]. In A. halophytica, by contrast, GB is synthesized from glycine by a three-step methylation reaction that is catalyzed by two N-methyltransferases (ApGSMT and ApDMT) [14,18]. ApGSMT is responsible for the first two methylation reactions that convert glycine to sarcosine and then convert sarcosine to dimethylglycine. ApDMT is responsible for the specific methylation of dimethylglycine to generate GB [14,18]. The cyanobacterium Synechocystis sp. PCC 6803 belongs to a group of intermediately salt-tolerant cyanobacteria [42]. It can grow at NaCl concentrations of up to 1.3 M. These cyanobacteria synthesize glucosylglycerol as a compatible solute via the actions of two enzymes, namely glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase. In some cases, they also synthesize sucrose as a compatible solute. A third group, of which Synechococcus elongates is a member, contains cyanobacteria that are less tolerant to salt stress then the cyanobacteria mentioned above [42]. S. elongates can grow at NaCl concentrations of up to 0.4 M. It synthesizes sucrose and/or trehalose as compatible solutes. In conclusion, for cyanobacteria, GB appears to be the most effective compatible solute for the toleration of extreme salt levels in their natural habitat.
machinery was protected against salt stress and cold stress more efficiently than it was in rice plants transformed with a cytosol-targeted codA, even though the transgenic plants of cytosol-targeted codA accumulated fivefold higher levels of GB in leaves [15]. A further three types of transgenic tomato plants were generated using a codA gene that was targeted to chloroplasts (Chl-codA plants), to the cytosol (Cyt-codA plants), or to both chloroplasts and cytosol simultaneously (ChlCytcodA plants) [20]. Cyt-codA and ChlCyt-codA plants accumulated up to 5.0- and 6.6-fold, respectively, higher levels of GB in their leaves than did Chl-codA plants (0.3 mmol g 1 FW). All three types of transgenic plants exhibited greater chilling tolerance than wild-type plants. In Chl-codA plants, the stress tolerance of photosystem II (PSII) and the frequency of seed germination were similar to those in the other two types of transgenic plant. However, the stress tolerance during the growth of seedlings of Chl-codA plants was higher than those of transgenic plants, even though the level of GB was the lowest in the former plants. These observations led to the conclusion that the accumulation of GB in chloroplasts is more effective than the accumulation of GB in the cytosol for the protection of plants against abiotic stress. There appeared to be a significant correlation between levels of GB in chloroplasts and the extent of tolerance to oxidative stress.
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By contrast, there was no evident correlation between the level of GB in the cytosol and the extent of stress tolerance. In betA-transformed maize plants, the total amount of GB in leaves was much higher than that in leaves of wildtype plants [16,17]. Levels of GB in the chloroplasts of both the transgenic and the wild-type plants were very low (0.1 mmol g 1 FW in both cases). Nonetheless, there was a significant difference in drought tolerance between these two types of plant [16]. Therefore, it seems likely that, in maize plants, the enhanced tolerance to drought stress resulted from the GB in the cytosol. The strong protective effect of GB in reproductive organs Plants are most sensitive to abiotic stress at the reproductive stage. Moreover, protection of plants against abiotic stress at the reproductive stage is essential for high yields in stress-prone areas around the world. It appears that introduction of the GB-biosynthetic pathway into crop plants is an effective strategy for increasing the yield of crop plants under abiotic stress. Analysis of levels of GB in codA-transgenic Arabidopsis plants revealed that the various organs accumulated different levels of GB, even though the codA gene was driven by a constitutive CaMV 35S promoter [9]. In fully matured plants, levels of GB in flowers, siliques and inflorescence apices were about fivefold higher than those in leaves. Such elevated levels of GB in the reproductive organs of the transgenic plants might have contributed to the increased numbers of flowers, siliques and seeds when the plants were grown under stress conditions. Transient exposure of wild-type plants to salt stress for three days resulted in the abortion of flower buds and reduced numbers of seeds per silique. Microscopic examination of floral structures revealed that, in wild-type plants (Figure 1a), salt stress inhibited the development of anthers, pistils and petals, and the production of pollen grains and ovules was dramatically inhibited. In GBaccumulating transgenic plants (Figure 1b), these effects of salt stress were significantly reduced. The transgenic plants produced 21% and 45% more siliques and seeds, respectively, than did wild-type plants after transient exposure of plants to 100 mM NaCl for three days [9]. These results revealed that the accumulation of GB in reproductive organs can effectively protect the formation of flowers and seeds against salt stress in GB-accumulating plants. Tomato plants are susceptible to chilling stress at the reproductive stage. The effects of chilling treatment at 3 8C for one week on mature plants were examined [5], and plants that harbored the codA transgene exhibited a significant increase in flower retention and in fruit set after exposure to such chilling conditions. Overall, the GBaccumulating transgenic tomato plants produced an average of 10% to 30% more fruits after the chilling treatment than did wild-type plants. When betA-transformed maize plants were exposed to drought stress in the field, it was found that much larger amounts of GB accumulated in leaves of transgenic plants than in leaves of wild-type plants [16,17]. When wild-type and transgenic plants were subjected to drought stress for 501
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Figure 1. The codA transgene in Arabidopsis thaliana enhanced the development of flower buds after salt stress. Wild-type and codA-transgenic plants were grown under normal conditions and then treated with 100 mM NaCl for three days. Plants were then allowed to grow under normal conditions (no additional NaCl in the growth medium) for a further six days. Micrographs show radial sections of flower buds from wild-type (a) and codA-transgenic (b) plants. Bars correspond to 0.2 mm. This figure was reproduced, with permission, from Ref. [8].
three weeks, the number of pollen grains fell and the formation of ears was delayed in both types of plant. However, reproductive development (as measured by the number of pollen grains and the silking time) of transgenic plants was less inhibited by drought than that of wild-type plants. After drought treatment for three weeks, the root biomass, plant height, stem biomass and leaf biomass of transgenic plants were all greater than those of wild-type plants. The weight of grains per plant in transgenic lines was 10% to 23% higher than that of wild-type plants due to an increase in both the number and weight of grains. Thus, transgenic maize plants, which accumulated elevated levels of GB, were less susceptible than wild-type plants to drought stress. Effect of GB on flowers, fruits and seeds under nonstress conditions We have examined the yield and quality of abiotic stresstolerant transgenic plants grown under both non-stress
and stressful conditions. Under non-stress conditions, the codA transgene had no negative effects on the germination of Arabidopsis seeds [21] and on the growth of young plants [22]. However, codA-transgenic Arabidopsis plants produced 22% more flowers (and siliques) and 28% more seeds than wild-type plants when grown under non-stress conditions [9]. When wild-type and codA-transgenic tomato plants were grown under normal conditions, there were no visible differences between the plants before flowering [23]. However, the presence of the codA transgene clearly resulted in increased sizes of flower buds and flowers under normal conditions (Figure 2a) [23]. Increases in the sizes of all floral organs were also evident. However, the number of petal segments was the same in wild-type and transgenic flowers. The ripe fruits of transgenic tomato plants were also considerably larger than those of wild-type plants (Figure 2b); the ripe fruits from transgenic plants were, on average, 54% heavier than those from wild-type plants
Figure 2. The codA transgene induced the formation of enlarged flowers and fruits on tomato plants under non-stress conditions. WT and codA represent wild-type and codA-transgenic plants, respectively. (a) Flower buds two days before anthesis (I) and flowers seven days after anthesis (II). (b) Overview and cross section of fruits. The bars correspond to 1.0 cm. This figure was reproduced, with permission, from Ref. [23].
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Review [23]. A microscopic examination of carpel and petal tissues revealed that the enlargement of flowers on codA-transgenic plants was related to the enhanced division of cells and the enhanced post-mitotic expansion of cells in the outer wall of the pericarp during the development of flower buds. Protection of the photosynthetic machinery In the 1990s, in vitro studies demonstrated that at concentrations as high as 1.0 M, GB stabilizes the oxygenevolving PSII complex against high concentrations of salts or high temperature by protecting PSII against the dissociation of the extrinsic polypeptides [4]. However, the concentrations of GB that are effective in the protection of transgenic plants against abiotic stress are of the order of mmol g 1 FW (Table 2). Even if GB is accumulated in chloroplasts exclusively, the concentration of GB is calculated to be less than 100 mM [24]. These findings suggest that the mechanism(s) operating in vivo differs from the simple stabilization of the PSII complex that can be observed in vitro. Abiotic stress, such as a high concentration of NaCl, might inactivate the translational machinery directly [25,26] and/or it might act indirectly via ROS, which are generated upon inhibition of the fixation of CO2 [27,28] and inhibit translation [29–33]. This latter hypothesis is based on the finding that Rubisco is inactivated under moderate heat stress [34,35] and also in the presence of a high concentration of NaCl [36]. In the case of indirect inactivation, the primary target of abiotic stress is postulated to be Rubisco or Rubisco activase. When the cyanobacterium Synechococcus sp. PCC 7942 (hereafter Synechococcus) was transformed with a codA gene, cells synthesized GB in the presence of exogenously supplied choline and accumulated GB at levels of 60 to 80 mM. GB accumulated in transgenic Synechococcus counteracted the inhibitory effect of salt stress on the repair of PSII but it did not affect the photodamage to PSII [37]. Pulse-chase radio-labeling experiments revealed that salt stress inhibited both the degradation of the D1 protein in photodamaged PSII and the synthesis of the D1 protein de novo, and it was also found that GB counteracted the salt-induced inhibition of the degradation and synthesis of D1. Neither salt stress nor the accumulation of GB affected levels of psbA transcripts [37]. These observations suggest that GB might counteract the inhibitory effects of salt stress on both translation and proteolysis, resulting in acceleration of the repair of photodamaged PSII under abiotic stress. Transgenic tobacco plants that had been transformed with the BADH gene for betaine aldehyde dehydrogenase from spinach accumulated 63% to 87% of the total GB in chloroplasts [35]. Analysis of these transgenic tobacco plants indicated that moderate heat stress, namely 40– 50 8C, stimulated the photoinhibition of PSII and that the accumulation of GB in vivo increased the resistance of PSII to heat-stress-stimulated photoinhibition [31]. The protective effect of GB on PSII was due to enhancement of the repair of PSII under moderate heat stress. It seems likely that inhibition of CO2 fixation under moderate heat stress might accelerate the generation of ROS, which, in turn,
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inhibit the synthesis of proteins, including the D1 protein [38]. Therefore, it is very likely that GB might stabilize Rubisco under moderate heat stress and thus decrease the generation of ROS, resulting in the protection of the translational machinery. By contrast, in cyanobacteria, which lack Rubisco activase, moderate heat stress seems to affect the translational machinery directly, and GB acts by counteracting this negative effect on the synthesis of the D1 protein [39]. It appears that the accumulation of GB in vivo enhances the thermotolerance of cyanobacteria by counteracting the inhibitory effects of moderate heat stress on the repair of photodamaged PSII. GB-induced expression of specific genes The effective concentrations of GB after uptake of exogenously applied GB or as a result of the genetically engineered synthesis of GB in vivo are in the millimolar range, as are the effective concentrations of plant hormones [40]. Thus, it is reasonable to postulate that the effects of GB might be manifested through the induction of expression of specific genes whose products are involved in the development of stress tolerance. The accumulation of three cold-responsive proteins after the exogenous application of GB to wheat plants was examined [2]. The extent of the accumulation of WCOR410 (wheat cold-regulated 410) protein was dependent on the concentration of GB, whereas the accumulation of the other two proteins, WCS120 (wheat cold-specific 120) and WCS413, was unaffected by the amount of applied GB. However, transcripts of both the wcor410 and wcor413 genes accumulated upon exogenous application of 250 mM GB. In tomato plants, application of exogenous GB resulted in enhanced catalase activity and an elevated level of the transcript of cat1 gene, and this effect was strongest one day after chilling treatment [10]. DNA microarray analysis was used to identify genes whose expression was enhanced by the exogenous application of 100 mM GB to both leaves and roots of Arabidopsis [40]. Genes whose expression was enhanced by GB included genes for transcription factors, for membranetrafficking components, for ROS-scavenging enzymes and for NADP-dependent ferric reductase that is located on the plasma membrane. Northern blot analysis revealed that the expression of four genes was significantly enhanced during treatment of roots with GB for 24 h. Wild-type plants and ‘knockout mutants’ that lacked an intact RabAc4 gene were treated either with or without 100 mM GB. After treatment with GB for 24 h, plants were subjected to chilling at 4 8C for a further 24 h and then incubated under warm conditions for four days. Wild-type plants responded to GB treatment with clearly enhanced growth of roots after the episode of chilling stress, whereas RabA4C-knockout plants were basically unaffected by the treatment with GB. The authors concluded that RabA4C is required for the effect of GB on chilling tolerance and that treatment with GB enhances stress tolerance, at least to some extent, via gene activation. A tomato cDNA microarray was used to compare gene expression in flower buds of wild-type and codA-transgenic tomato plants [23]. The expression of 30 genes was 503
Review enhanced and that of 29 genes was repressed in the flower buds of the codA-transgenic plants as compared with wildtype plants. Among the genes whose expression was enhanced, several are known to be involved in cell division, such as the genes for cyclin D (CycD3-1), cyclin-dependent protein kinase p34/cdc2 (CDKA1) and related-to-ubiquitinconjugating enzyme (RCE1). In addition, analysis by semiquantitative reverse transcription (RT)-PCR revealed that GB repressed the expression of the fw2.2 gene, whose product acts as a negative regulator of cell division in the early stages of fruit development [41] in flowers but not in young leaves and flower buds. Taken together, the various observations discussed above indicate that GB, either applied exogenously or synthesized in transgenic plants, seems to be capable of activating specific genes. Identification of GB-inducible genes and the functions of their products will advance our understanding of GB-enhanced stress tolerance in plants. Conclusion and perspectives A variety of genes has been employed to generate transgenic plants that accumulate GB and are tolerant to abiotic stress. Among the properties of these transgenic plants, the physiological and morphological aspects of stress tolerance have been investigated most extensively in codAtransgenic plants. Studies have focused on stress tolerance at all stages of the life cycle of plants, from imbibition of seeds, through the growth of young plants and the photosynthetic activity of mature plants, to the production of fruits and seeds. The protective effects of GB in the reproductive organs of plants have been observed in Arabidopsis, maize and tomato. This protection might have been mediated by the high levels of GB that accumulate in reproductive organs as a consequence of the translocation of GB from other organs, such as leaves. In addition, the codA transgene enhances the productivity of seeds and increases the size of fruits and flowers under non-stress conditions. Further studies should allow the enhancement of stress tolerance in numerous crop species, as well as increases in yield. Possible mechanisms for the GB-enhanced tolerance of plants to various types of abiotic stress include, but are not limited to: (i) protection of the photosynthetic machinery; (ii) induction of specific genes whose products are involved in stress tolerance; (iii) reductions in levels of ROS under stress; and (iv) regulation of the activities of ion-channel proteins either directly or via protection of the plasma membrane. References 1 Rhodes, D. and Hanson, A.D. (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384 2 Allard, F. et al. (1998) Betaine improves freezing tolerance in wheat. Plant Cell Physiol. 39, 1194–1202 3 Kishitani, S. et al. (1994) Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants. Plant Cell Environ. 17, 89–95 4 Papageorgiou, G.C. and Murata, N. (1995) The unusually strong stabilizing effects of glycinebetaine on the structure and function of the oxygen-evolving photosystem II complex. Photosynth. Res. 44, 243–252
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Trends in Plant Science Vol.13 No.9 5 Sakamoto, A. and Murata, N. (2000) Genetic engineering of glycinebetaine synthesis in plants: current status and implication for enhancement of stress tolerance. J. Exp. Bot. 51, 81–88 6 Sakamoto, A. and Murata, N. (2001) The use of choline oxidase, a glycinebetaine-synthesizing enzyme, to create stress resistant transgenic plants. Plant Physiol. 125, 180–188 7 Sakamoto, A. and Murata, N. (2002) The role of glycinebetaine in the protection of plants from stress: clues from transgenic plants. Plant Cell Environ. 25, 163–171 8 Sulpice, R. et al. (2003) Enhanced formation of flowers in salt-stressed Arabidopsis after genetic engineering of the synthesis of glycinebetaine. Plant J. 36, 165–176 9 Park, E.J. et al. (2004) Genetic engineering of glycinebetaine synthesis in tomato protects seeds, plants, and flowers from chilling damage. Plant J. 40, 474–487 10 Park, E.J. et al. (2006) Exogenous application of glycinebetaine increases chilling tolerance in tomato plants. Plant Cell Physiol. 47, 706–714 11 Park, E.J. et al. (2003) Genetic engineering of cold-tolerant tomato via glycinebetaine biosynthesis. Cryobiol. Cryotech. 49, 77–85 12 Ma¨kela¨, P. et al. (1996) Uptake and translocation of foliar-applied glycinebetaine in crop plants. Plant Sci. 121, 221–230 13 Cuin, T.A. and Shabala, S. (2007) Compatible solutes reduce ROSinduced potassium efflux in Arabidopsis roots. Plant Cell Environ. 30, 875–885 14 Chen, T.H.H. and Murata, N. (2002) Enhancement of tolerance to abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr. Opin. Plant Biol. 5, 250–257 15 Sakamoto, A. et al. (1998) Metabolic engineering of rice leading to biosynthesis of glycinebetaine and tolerance to salt and cold. Plant Mol. Biol. 38, 1011–1019 16 Quan, R. et al. (2004) Improved chilling tolerance by transformation with betA gene for the enhancement of glycinebetaine synthesis in maize. Plant Sci. 166, 141–149 17 Quan, R. et al. (2004) Engineering of enhanced glycinebetaine synthesis improves drought tolerance in maize. Plant Biotechnol. J. 2, 477–486 18 Waditee, R. et al. (2005) Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc. Natl. Acad. Sci. U. S. A. 102, 1318–1323 19 Su, J. et al. (2006) Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stressprotectant glycinebetaine. J. Exp. Bot. 57, 1129–1135 20 Park, E.J. et al. (2007) Glycinebetaine accumulation in chloroplasts is more effective than that in cytosol in protecting transgenic tomato plants against abiotic stress. Plant Cell Environ. 30, 994–1005 21 Alia et al. (1998) Transformation with a gene for choline oxidase enhances the cold tolerance of Arabidopsis during germination and early growth. Plant Cell Environ. 21, 232–239 22 Alia et al. (1998) Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine. Plant J. 16, 155–161 23 Park, E.J. et al. (2007) The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato. Plant Biotechnol. J. 5, 422–430 24 Hayashi, H. et al. (1997) Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J. 12, 133–142 25 Brady, C.J. et al. (1984) Salt tolerance in plants. I. Ions, compatible organic solutes and the stability of plant ribosomes. Plant Cell Environ. 7, 571–578 26 Gibson, T.S. et al. (1984) Salt tolerance in plants. II. In vitro translation of mRNAs from salt-tolerant and salt-sensitive plants on wheat germ ribosomes: responses to ions and compatible organic solutes. Plant Cell Environ. 7, 579–587 27 Murata, N. et al. (2007) Photoinhibition of photosystem II under environmental stress. Biochim. Biophys. Acta (Bioenergetics) 1767, 414–421 28 Takahashi, S. and Murata, N. (2008) How do environmental stresses stimulate photoinhibition? Trends Plant Sci. 13, 178–182 29 Nishiyama, Y. et al. (2004) Singlet oxygen inhibits the repair of photosystem II by suppressing the translation elongation of the
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D1 protein in Synechocystis sp. PCC 6803. Biochemistry 43, 11321– 11330 Al-Taweel, K. et al. (2007) A bacterial transgene for catalase protects translation of D1 protein during exposure of salt-stressed tobacco leaves to strong light. Plant Physiol. 145, 258–265 Yang, X. et al. (2007) Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants. Planta 225, 719–733 Nishiyama, Y. et al. (2005) Inhibition of the repair of photosystem II by oxidative stress in cyanobacteria. Photosynth. Res. 84, 1–7 Nishiyama, Y. et al. (2006) A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II. Biochim. Biophys. Acta (Bioenergetics) 1757, 742–749 Feller, U. et al. (1998) Moderately high temperatures inhibit ribulose1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase-mediated activation of Rubisco. Plant Physiol. 116, 539–546 Yang, X. et al. (2005) Genetic engineering of the biosynthesis of glycinebetaine enhances photosynthesis against high temperature stress in transgenic tobacco plants. Plant Physiol. 138, 2299–2309 Solomon, A. et al. (1994) Effects of NaCl on the carboxylating activity of Rubisco from Tamarix jordanis in the presence and absence of prolinerelated compatible solutes. Physiol. Plant. 90, 198–204 Ohnishi, N. and Murata, N. (2006) Glycinebetaine counteracts the inhibitory effects of salt stress on the degradation and synthesis of the D1 protein during photoinhibition in Synechococcus sp. PCC 7942. Plant Physiol. 141, 756–765 Takahashi, S. and Murata, N. (2006) Glycerate-3-phosphate, produced by CO2 fixation in the Calvin cycle, is critical for the synthesis of the D1 protein of photosystem II. Biochim. Biophys. Acta (Bioenergetics) 1757, 198–205 Allakhverdiev, S.I. et al. (2007) Glycinebetaine alleviates the inhibitory effect of moderate heat stress on the repair of photosystem II during photoinhibition. Biochim Biophys. Acta (Bioenergetics) 1767, 1363– 1371 Einset, J. et al. (2007) Membrane-trafficking RabA4c involved in the effect of glycine betaine on recovery from chilling stress in Arabidopsis. Physiol. Plant 130, 511–518 Frary, A. et al. (2000) Cloning and transgenic expression of fw2.2: a quantitative trait locus key to the evolution of tomato fruit. Science 289, 85–87 Joset, F. et al. (1996) Dynamics of the response of cyanobacteria to salt stress: deciphering the molecular events. Physiol. Plant. 96, 738–744 Ishitani, M. et al. (1993) Regulation of glycinebetaine accumulation in the halotolerant cyanobacterium Aphanothece halophytica. Aust. J. Plant Physiol. 20, 693–703 Hayashi, H. et al. (1998) Enhanced germination under high-salt conditions of seeds of transgenic Arabidopsis with a bacterial gene (codA) for choline oxidase. J. Plant Res. 111, 357–362 Xing, W. and Rajashekar, C.B. (2001) Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana. Environ. Exp. Bot. 46, 21–28 Sakamoto, A. et al. (2000) Transformation of Arabidopsis with the codA gene for choline oxidase enhances freezing tolerance of plants. Plant J. 22, 449–453
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47 Ma¨kela¨, P. et al. (1999) Photosynthetic response of drought- and saltstressed tomato and turnip rape plants to foliar-applied glycinebetaine. Physiol. Plant. 105, 45–50 48 Rajashekar, C.B. et al. (1999) Glycine betaine accumulation and induction of cold tolerance in strawberry (Fragaria X ananassa Duch.) plants. Plant Sci. 148, 175–183 49 Jokinen, K. et al. (1999) Glycinebetaine from sugar beet enhances the yield of field-grown tomatoes. Acta Hortic. 487, 233–236 50 Ma¨kela¨, P. et al. (1998) Foliar application of glycinebetaine – a novel product from sugar beet – as an approach to increase tomato yield. Ind. Crops Prod. 7, 139–148 51 Zhao, Y. et al. (1992) Protection of membrane integrity in Medicago sativa L. by glycinebetaine against the effects of freezing. J. Plant Physiol. 140, 541–543 52 Harinasult, P. et al. (1996) Exogenous glycinebetaine accumulation and increased salt-tolerance in rice seedlings. Biosci. Biotechnol. Biochem. 60, 366–368 53 Xing, W. and Rajashekar, C.B. (1999) Alleviation of water stress in beans by exogenous glycine betaine. Plant Sci. 148, 185–195 54 Chen, W.P. et al. (2000) Glycinebetaine increases chilling tolerance and reduces chilling-induced lipid peroxidation in Zea mays L. Plant Cell Environ. 23, 609–618 55 Alia et al. (1999) Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase. Plant Mol. Biol. 40, 279–288 56 Huang, J. et al. (2000) Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: metabolic limitations. Plant Physiol. 122, 747–756 57 Hibino, T. et al. (2002) Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants. J. Biol. Chem. 277, 41352–41360 58 Prasad, K.V.S.K. et al. (2000) Transformation of Brassica juncea (L.) Czern with a bacterial codA gene enhances its tolerance to salt stress. Mol. Breed. 6, 489–499 59 Gao, M. et al. (2000) Transformation of Japanese persimmon (Diospyros kaki Thunb.) with a bacterial gene for choline oxidase. Mol. Breed. 6, 501–510 60 Lv, S. et al. (2007) Increase of glycinebetaine synthesis improves drought tolerance in cotton. Mol. Breed. 20, 233–248 61 Holmstro¨m, K-O. et al. (2000) Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J. Exp. Bot. 51, 177–185 62 Takabe, T. et al. (1998) Evaluation of glycinebetaine accumulation for stress tolerance in transgenic rice plants. In Proceedings of International Workshop on Breeding and Biotechnology for Environmental Stress in Rice: Sapporo, Japan, pp. 63–68 63 Shirasawa, K. et al. (2006) Accumulation of glycinebetaine in rice plants that overexpress choline monooxygenase from spinach and evaluation of their tolerance to abiotic stress. Ann. Bot. (Lond.) 98, 565–571 64 Mohanty, A. et al. (2002) Transgenics of an elite indica rice variety Pusa Basmati 1 harboring the codA gene are highly tolerant to salt stress. Theor. Appl. Genet. 106, 51–57 65 Ahmad, R. et al. (2008) Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses. Plant Cell Rep. 27, 687–698
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Glycinebetaine: an effective protectant against abiotic stress in plants Tony H.H. Chen1 and Norio Murata2 1 2
Department of Horticulture, ALS 4017, Oregon State University, Corvallis, OR 97331, USA National Institute for Basic Biology, Okazaki 444-8585, Japan
Glycinebetaine (GB) has been studied extensively as a compatible solute because of the availability of GBaccumulating transgenic plants that harbor a variety of transgenes for GB-biosynthetic enzymes. Both the exogenous application of GB and the genetically engineered biosynthesis of GB increase the tolerance of plants to abiotic stress. As reviewed here, studies of such increased tolerance to abiotic stress have led to considerable progress in the characterization of the roles of GB in stress tolerance in plants. In particular, the reproductive organs of GB-accumulating transgenic plants exhibit enhanced tolerance to abiotic stress. Furthermore, accumulation of GB results in increased yield potentials under non-stress conditions. Glycinebetaine (GB), an effective protectant against abiotic stress GB is a fully N-methyl-substituted derivative of glycine, and it is found in a large variety of microorganisms, higher plants and animals [1]. GB belongs to a group of compounds that are known collectively as ‘compatible solutes’: small organic metabolites that are very soluble in water and non-toxic at high concentrations. GB accumulates in the chloroplasts and plastids of many halotolerant plants. These plants accumulate high levels of GB in response to abiotic stress [2,3]. Levels of accumulated GB are generally correlated with the extent of stress tolerance [1]. GB effectively stabilizes the quaternary structures of enzymes and complex proteins, and it maintains the highly ordered state of membranes at non-physiological temperatures and salt concentrations [4]. Both the exogenous application of GB (Table 1) and the introduction, via transgenes, of the GB-biosynthetic pathway into plants that do not naturally accumulate GB (Table 2) increase the tolerance of such plants to various types of abiotic stress. This increased tolerance to abiotic stress provides useful systems for investigating the mechanisms by which GB protects plants against abiotic stress. Among the compatible solutes, GB is a particularly effective protectant against abiotic stress [5–7]. Studies of GB have focused on GB-mediated tolerance to various kinds of stress and at various stages of the life cycle of plants [8,9]. Exogenous application of GB Exogenous application of GB can improve the tolerance of numerous plant species to various types of abiotic stress, and it can enhance subsequent growth and yield (Table 1). Corresponding author: Murata, N. ([email protected]).
When applied to leaves of plants, GB is readily taken up by leaf tissues [10]. GB can also be taken up via roots [11]. When GB is applied to the leaves of tomato (Lycopersicon esculentum) plants, most of the GB that is taken up by the leaves is localized in the cytosol and only a small fraction of the cytosolic GB is translocated to chloroplasts [10]. When [14C]glycinebetaine was applied to leaves of summer turnip (Brassica rapa L.) plants, the radio-labeled GB was translocated to roots within two hours [11]. During the next 24 h, the radio-labeled GB was translocated to all parts of the plant. In tomato plants, large amounts of foliar-applied GB were translocated to meristem-containing tissues, including the flower buds and shoot apices. It was demonstrated that GB was translocated to actively growing and expanding portions of plants, the long-distance translocation of GB being mediated by the phloem [12]. Reactive oxygen species (ROS) are produced continuously as products of various metabolic pathways, even when plants are growing under non-stress conditions. These ROS are scavenged by a variety of antioxidant defense systems that prevent ROS from reaching toxic levels. All forms of abiotic stress, including high salt, chilling, freezing and drought stress, cause an oxidative burst in plant cells. The application of hydroxyl radicals (OH*) to Arabidopsis roots resulted in a massive, dosedependent efflux of K+ ions from epidermal cells in the elongation zone [13]. However, the presence of GB at 5 mM in the incubation medium significantly reduced this efflux of K+ ions. Furthermore, in tomato plants, exogenously applied GB significantly reduced the chilling-induced production of H2O2 [10]. Because GB does not scavenge ROS directly, GB must mitigate the damaging effects of oxidative stress in other ways, for example by activating or stabilizing ROS-scavenging enzymes and/or repressing the production of ROS by an unknown mechanism. Genetically engineered biosynthesis of GB GB is synthesized either by the oxidation (or dehydrogenation) of choline or by the N-methylation of glycine [14]. Genes that encode GB-biosynthetic enzymes have been cloned from various microorganisms and plants, and transgenic plants of various species have been produced that express one or several of these genes (Table 2). The transgenic plants accumulate GB at a variety of levels and exhibit enhanced tolerance to a variety of abiotic stresses (Table 2). In plants that do not accumulate GB naturally, the highest levels of accumulated GB have been found in leaves of codA-transgenic rice (Oryza sativum) plants
1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.06.007 Available online 14 August 2008
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Table 1. Enhancement of tolerance to abiotic stress via exogenously applied glycinebetaine in higher plants Plant species Arabidopsis thaliana
Avena sativa Brassica rapa Fragaria X ananassa Hordeum vulgare Lycopersicon esculentum
Medicago sativa Oryza sativa Phaseolus vulgaris Triticum aestivum Zea mays
Types of stress Salt Freezing Freezing Oxidative Chilling Drought, salt Drought, salt Drought, salt Freezing Drought, salt Chilling Drought Drought, salt Drought, salt Freezing Salt Drought Freezing Drought, salt Chilling
Environment
Refs
Growth chamber Greenhouse Growth chamber Growth chamber Growth chamber Greenhouse, field Greenhouse Greenhouse, field Greenhouse Greenhouse, field Growth chamber Field Growth chamber Greenhouse Growth chamber Growth chamber Greenhouse Growth chamber Greenhouse, field Growth chamber
[44] [45] [46] [13] [40] [12] [47] [12] [48] [12] [5,10,11] [49] [50] [47] [51] [52] [53] [2] [12] [54]
[5.3 mmol g 1 fresh weight (FW)] [15]. In a natural GBaccumulator, maize (Zea mays), the highest reported level of GB in leaves of betA-transgenic maize plants is 5.7 mmol g 1 FW [16,17]. Thus, in terms of maximum levels of GB in transgenic plants, there appears to be no significant difference between plants that accumulate GB naturally and those that do not. Furthermore, glycinemethylation enzymes, such as ApGSMT and ApDMT (Aphanothece halophytica glycine sarcosine methyltrans-
ferase and A. halophytica dimethylglycine methyltransferase, respectively; see Box 1), do not have any apparent advantages over choline-oxidizing enzymes in efforts to engineer the biosynthesis of GB in plants [18]. GB-producing transgenic rice lines were generated in which the cox gene for choline oxidase from Arthrobacter pascens, fused to a chloroplast-targeting sequence, was expressed under the control of either an abscisic acid (ABA)-inducible promoter [a stress-inducible promoter (SIP)] or the promoter of a constitutively expressed gene for ubiquitin (UBI) [19]. The highest level of GB that accumulated under high-NaCl conditions [2.60 mmol g 1 dry weight (DW)] in SIP lines was not as high as that in UBI lines (3.12 mmol g 1 DW). Therefore, the use of an ABA-inducible promoter was not particularly effective for production of GB de novo. Accumulation of GB in chloroplasts Two major factors that influence the tolerance of plants to abiotic stress are the concentration and the localization of GB in the cell. In many studies of the engineered accumulation of GB in plants, GB-biosynthetic enzymes have been targeted to chloroplasts. In a few studies, the enzymes have been targeted to either the cytosol or the mitochondria, or to both the cytosol and the chloroplasts simultaneously (Table 2). In codA-transgenic plants, the gene product has been targeted predominantly to chloroplasts [14]. The resultant transgenic plants accumulate GB primarily in their chloroplasts, and they exhibit tolerance to various kinds of abiotic stress [5–9]. In rice plants transformed with a chloroplast-targeted codA construct, the photosynthetic
Table 2. Transgenic plants engineered to synthesize glycinebetaine and their enhanced tolerance to abiotic stress Plant species Arabidopsis thaliana
Gene codA codA codA codA codA codA cox CMO +BADH ApGSMT +ApDMT cox Brassica napus codA Brassica juncea codA Diospyras kaki betA Gossypium hirsutum Lycopersicon esculentum coda
Nicotiana tabacum
Oryza sativa
Solanum tuberosum Zea mays
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Subcellular location Chloroplasts Chloroplasts Chloroplasts Chloroplasts Chloroplasts Chloroplasts Cytosol Unknown Cytosol
Maximum accumulation 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.2 mmol g 1 FW 1.9 mmol g 1 DW 900 mmol g 1 DW 2.0 mmol g 1 FW [+5 mM glycine] Cytosol 13 mmol g 1 DW Chloroplasts 0.82 mmol g 1 FW Chloroplasts 0.3 mmol g 1 FW Cytosol 354.3 mmol g 1 DW Chloroplasts 0.3 [leaf] mmol g 1 FW 1.2 [Flower] mmol g 1 FW Chloroplasts 23 nmol mg 1 chlorophyll codA Cytosol 95 nmol mg 1 chlorophyll codA Chloroplasts + cytosol 120 nmol mg 1 chlorophyll codA cox Cytosol 13 mmol g 1 DW Cytosol 35 nmol g 1 FW betA Chloroplasts 4.6 mmol g 1 FW BADH coda Cytosol 5.3 mmol g 1 FW Chloroplasts 1.1 mmol g 1 FW codA 5.1 mmol g 1 FW betA (modified) Mitochondria Chloroplasts 3.1 mmol g 1 DW cox Chloroplasts 0.43 mmol g 1 DW CMO Chloroplasts 2.12 mmol g 1 DW codA codA Chloroplasts 1.43 mmol g 1 FW betA Cytosol 5.7 mmol g 1 FW Cytosol 5.7 mmol g 1 FW betA
Enhanced tolerance Chilling Chilling, salt Heat Strong light Freezing Salt Salt, drought, freezing Salt Salt, chilling CuSO4 Drought, salt Salt Salt Drought Chilling Salt Chilling, salt Oxidative Salt Salt, chilling Heat Salt, chilling Salt, chilling Salt, drought Salt Salt, day/night temperature (28/13 8C) Salt Oxidative, salt, drought Chilling Drought
Refs [21] [24] [22] [55] [46] [8,44] [56] [57] [18] [56] [58] [59] [60] [9] [20]
[56] [61] [31,35] [15] [15] [62] [19] [63] [64] [65] [16] [17]
Review Box 1. Accumulation of glycinebetaine (GB) in the halotolerant cyanobacterium Aphanothece halophytica Cyanobacteria are prokaryotes with photosynthetic machinery that resembles that of plants both structurally and functionally [42], and these cyanobacteria are capable of oxygenic photosynthesis. Cyanobacteria have successfully colonized a wide range of environments. Several strains of cyanobacteria grow naturally in hypersaline water, such as the Dead Sea and the Solar Lake in the Sinai Desert [42]. The most halotolerant species can grow in concentrations of NaCl as high as 3 M [42,43]. These cyanobacteria are extremely tolerant to salt stress and synthesize and accumulate GB in their cytoplasm [42,43]. The sustained growth under salt stress of Aphanothece halophytica, an extremely salt-tolerant cyanobacterium isolated from the Dead Sea, is mediated by the accumulation of GB [42,43]. GB is the major compatible solute in this cyanobacterium [43]. Most known biosynthetic pathways to GB include the twostep oxidation of choline [14]. In A. halophytica, by contrast, GB is synthesized from glycine by a three-step methylation reaction that is catalyzed by two N-methyltransferases (ApGSMT and ApDMT) [14,18]. ApGSMT is responsible for the first two methylation reactions that convert glycine to sarcosine and then convert sarcosine to dimethylglycine. ApDMT is responsible for the specific methylation of dimethylglycine to generate GB [14,18]. The cyanobacterium Synechocystis sp. PCC 6803 belongs to a group of intermediately salt-tolerant cyanobacteria [42]. It can grow at NaCl concentrations of up to 1.3 M. These cyanobacteria synthesize glucosylglycerol as a compatible solute via the actions of two enzymes, namely glucosylglycerol phosphate synthase and glucosylglycerol phosphate phosphatase. In some cases, they also synthesize sucrose as a compatible solute. A third group, of which Synechococcus elongates is a member, contains cyanobacteria that are less tolerant to salt stress then the cyanobacteria mentioned above [42]. S. elongates can grow at NaCl concentrations of up to 0.4 M. It synthesizes sucrose and/or trehalose as compatible solutes. In conclusion, for cyanobacteria, GB appears to be the most effective compatible solute for the toleration of extreme salt levels in their natural habitat.
machinery was protected against salt stress and cold stress more efficiently than it was in rice plants transformed with a cytosol-targeted codA, even though the transgenic plants of cytosol-targeted codA accumulated fivefold higher levels of GB in leaves [15]. A further three types of transgenic tomato plants were generated using a codA gene that was targeted to chloroplasts (Chl-codA plants), to the cytosol (Cyt-codA plants), or to both chloroplasts and cytosol simultaneously (ChlCytcodA plants) [20]. Cyt-codA and ChlCyt-codA plants accumulated up to 5.0- and 6.6-fold, respectively, higher levels of GB in their leaves than did Chl-codA plants (0.3 mmol g 1 FW). All three types of transgenic plants exhibited greater chilling tolerance than wild-type plants. In Chl-codA plants, the stress tolerance of photosystem II (PSII) and the frequency of seed germination were similar to those in the other two types of transgenic plant. However, the stress tolerance during the growth of seedlings of Chl-codA plants was higher than those of transgenic plants, even though the level of GB was the lowest in the former plants. These observations led to the conclusion that the accumulation of GB in chloroplasts is more effective than the accumulation of GB in the cytosol for the protection of plants against abiotic stress. There appeared to be a significant correlation between levels of GB in chloroplasts and the extent of tolerance to oxidative stress.
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By contrast, there was no evident correlation between the level of GB in the cytosol and the extent of stress tolerance. In betA-transformed maize plants, the total amount of GB in leaves was much higher than that in leaves of wildtype plants [16,17]. Levels of GB in the chloroplasts of both the transgenic and the wild-type plants were very low (0.1 mmol g 1 FW in both cases). Nonetheless, there was a significant difference in drought tolerance between these two types of plant [16]. Therefore, it seems likely that, in maize plants, the enhanced tolerance to drought stress resulted from the GB in the cytosol. The strong protective effect of GB in reproductive organs Plants are most sensitive to abiotic stress at the reproductive stage. Moreover, protection of plants against abiotic stress at the reproductive stage is essential for high yields in stress-prone areas around the world. It appears that introduction of the GB-biosynthetic pathway into crop plants is an effective strategy for increasing the yield of crop plants under abiotic stress. Analysis of levels of GB in codA-transgenic Arabidopsis plants revealed that the various organs accumulated different levels of GB, even though the codA gene was driven by a constitutive CaMV 35S promoter [9]. In fully matured plants, levels of GB in flowers, siliques and inflorescence apices were about fivefold higher than those in leaves. Such elevated levels of GB in the reproductive organs of the transgenic plants might have contributed to the increased numbers of flowers, siliques and seeds when the plants were grown under stress conditions. Transient exposure of wild-type plants to salt stress for three days resulted in the abortion of flower buds and reduced numbers of seeds per silique. Microscopic examination of floral structures revealed that, in wild-type plants (Figure 1a), salt stress inhibited the development of anthers, pistils and petals, and the production of pollen grains and ovules was dramatically inhibited. In GBaccumulating transgenic plants (Figure 1b), these effects of salt stress were significantly reduced. The transgenic plants produced 21% and 45% more siliques and seeds, respectively, than did wild-type plants after transient exposure of plants to 100 mM NaCl for three days [9]. These results revealed that the accumulation of GB in reproductive organs can effectively protect the formation of flowers and seeds against salt stress in GB-accumulating plants. Tomato plants are susceptible to chilling stress at the reproductive stage. The effects of chilling treatment at 3 8C for one week on mature plants were examined [5], and plants that harbored the codA transgene exhibited a significant increase in flower retention and in fruit set after exposure to such chilling conditions. Overall, the GBaccumulating transgenic tomato plants produced an average of 10% to 30% more fruits after the chilling treatment than did wild-type plants. When betA-transformed maize plants were exposed to drought stress in the field, it was found that much larger amounts of GB accumulated in leaves of transgenic plants than in leaves of wild-type plants [16,17]. When wild-type and transgenic plants were subjected to drought stress for 501
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Figure 1. The codA transgene in Arabidopsis thaliana enhanced the development of flower buds after salt stress. Wild-type and codA-transgenic plants were grown under normal conditions and then treated with 100 mM NaCl for three days. Plants were then allowed to grow under normal conditions (no additional NaCl in the growth medium) for a further six days. Micrographs show radial sections of flower buds from wild-type (a) and codA-transgenic (b) plants. Bars correspond to 0.2 mm. This figure was reproduced, with permission, from Ref. [8].
three weeks, the number of pollen grains fell and the formation of ears was delayed in both types of plant. However, reproductive development (as measured by the number of pollen grains and the silking time) of transgenic plants was less inhibited by drought than that of wild-type plants. After drought treatment for three weeks, the root biomass, plant height, stem biomass and leaf biomass of transgenic plants were all greater than those of wild-type plants. The weight of grains per plant in transgenic lines was 10% to 23% higher than that of wild-type plants due to an increase in both the number and weight of grains. Thus, transgenic maize plants, which accumulated elevated levels of GB, were less susceptible than wild-type plants to drought stress. Effect of GB on flowers, fruits and seeds under nonstress conditions We have examined the yield and quality of abiotic stresstolerant transgenic plants grown under both non-stress
and stressful conditions. Under non-stress conditions, the codA transgene had no negative effects on the germination of Arabidopsis seeds [21] and on the growth of young plants [22]. However, codA-transgenic Arabidopsis plants produced 22% more flowers (and siliques) and 28% more seeds than wild-type plants when grown under non-stress conditions [9]. When wild-type and codA-transgenic tomato plants were grown under normal conditions, there were no visible differences between the plants before flowering [23]. However, the presence of the codA transgene clearly resulted in increased sizes of flower buds and flowers under normal conditions (Figure 2a) [23]. Increases in the sizes of all floral organs were also evident. However, the number of petal segments was the same in wild-type and transgenic flowers. The ripe fruits of transgenic tomato plants were also considerably larger than those of wild-type plants (Figure 2b); the ripe fruits from transgenic plants were, on average, 54% heavier than those from wild-type plants
Figure 2. The codA transgene induced the formation of enlarged flowers and fruits on tomato plants under non-stress conditions. WT and codA represent wild-type and codA-transgenic plants, respectively. (a) Flower buds two days before anthesis (I) and flowers seven days after anthesis (II). (b) Overview and cross section of fruits. The bars correspond to 1.0 cm. This figure was reproduced, with permission, from Ref. [23].
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Review [23]. A microscopic examination of carpel and petal tissues revealed that the enlargement of flowers on codA-transgenic plants was related to the enhanced division of cells and the enhanced post-mitotic expansion of cells in the outer wall of the pericarp during the development of flower buds. Protection of the photosynthetic machinery In the 1990s, in vitro studies demonstrated that at concentrations as high as 1.0 M, GB stabilizes the oxygenevolving PSII complex against high concentrations of salts or high temperature by protecting PSII against the dissociation of the extrinsic polypeptides [4]. However, the concentrations of GB that are effective in the protection of transgenic plants against abiotic stress are of the order of mmol g 1 FW (Table 2). Even if GB is accumulated in chloroplasts exclusively, the concentration of GB is calculated to be less than 100 mM [24]. These findings suggest that the mechanism(s) operating in vivo differs from the simple stabilization of the PSII complex that can be observed in vitro. Abiotic stress, such as a high concentration of NaCl, might inactivate the translational machinery directly [25,26] and/or it might act indirectly via ROS, which are generated upon inhibition of the fixation of CO2 [27,28] and inhibit translation [29–33]. This latter hypothesis is based on the finding that Rubisco is inactivated under moderate heat stress [34,35] and also in the presence of a high concentration of NaCl [36]. In the case of indirect inactivation, the primary target of abiotic stress is postulated to be Rubisco or Rubisco activase. When the cyanobacterium Synechococcus sp. PCC 7942 (hereafter Synechococcus) was transformed with a codA gene, cells synthesized GB in the presence of exogenously supplied choline and accumulated GB at levels of 60 to 80 mM. GB accumulated in transgenic Synechococcus counteracted the inhibitory effect of salt stress on the repair of PSII but it did not affect the photodamage to PSII [37]. Pulse-chase radio-labeling experiments revealed that salt stress inhibited both the degradation of the D1 protein in photodamaged PSII and the synthesis of the D1 protein de novo, and it was also found that GB counteracted the salt-induced inhibition of the degradation and synthesis of D1. Neither salt stress nor the accumulation of GB affected levels of psbA transcripts [37]. These observations suggest that GB might counteract the inhibitory effects of salt stress on both translation and proteolysis, resulting in acceleration of the repair of photodamaged PSII under abiotic stress. Transgenic tobacco plants that had been transformed with the BADH gene for betaine aldehyde dehydrogenase from spinach accumulated 63% to 87% of the total GB in chloroplasts [35]. Analysis of these transgenic tobacco plants indicated that moderate heat stress, namely 40– 50 8C, stimulated the photoinhibition of PSII and that the accumulation of GB in vivo increased the resistance of PSII to heat-stress-stimulated photoinhibition [31]. The protective effect of GB on PSII was due to enhancement of the repair of PSII under moderate heat stress. It seems likely that inhibition of CO2 fixation under moderate heat stress might accelerate the generation of ROS, which, in turn,
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inhibit the synthesis of proteins, including the D1 protein [38]. Therefore, it is very likely that GB might stabilize Rubisco under moderate heat stress and thus decrease the generation of ROS, resulting in the protection of the translational machinery. By contrast, in cyanobacteria, which lack Rubisco activase, moderate heat stress seems to affect the translational machinery directly, and GB acts by counteracting this negative effect on the synthesis of the D1 protein [39]. It appears that the accumulation of GB in vivo enhances the thermotolerance of cyanobacteria by counteracting the inhibitory effects of moderate heat stress on the repair of photodamaged PSII. GB-induced expression of specific genes The effective concentrations of GB after uptake of exogenously applied GB or as a result of the genetically engineered synthesis of GB in vivo are in the millimolar range, as are the effective concentrations of plant hormones [40]. Thus, it is reasonable to postulate that the effects of GB might be manifested through the induction of expression of specific genes whose products are involved in the development of stress tolerance. The accumulation of three cold-responsive proteins after the exogenous application of GB to wheat plants was examined [2]. The extent of the accumulation of WCOR410 (wheat cold-regulated 410) protein was dependent on the concentration of GB, whereas the accumulation of the other two proteins, WCS120 (wheat cold-specific 120) and WCS413, was unaffected by the amount of applied GB. However, transcripts of both the wcor410 and wcor413 genes accumulated upon exogenous application of 250 mM GB. In tomato plants, application of exogenous GB resulted in enhanced catalase activity and an elevated level of the transcript of cat1 gene, and this effect was strongest one day after chilling treatment [10]. DNA microarray analysis was used to identify genes whose expression was enhanced by the exogenous application of 100 mM GB to both leaves and roots of Arabidopsis [40]. Genes whose expression was enhanced by GB included genes for transcription factors, for membranetrafficking components, for ROS-scavenging enzymes and for NADP-dependent ferric reductase that is located on the plasma membrane. Northern blot analysis revealed that the expression of four genes was significantly enhanced during treatment of roots with GB for 24 h. Wild-type plants and ‘knockout mutants’ that lacked an intact RabAc4 gene were treated either with or without 100 mM GB. After treatment with GB for 24 h, plants were subjected to chilling at 4 8C for a further 24 h and then incubated under warm conditions for four days. Wild-type plants responded to GB treatment with clearly enhanced growth of roots after the episode of chilling stress, whereas RabA4C-knockout plants were basically unaffected by the treatment with GB. The authors concluded that RabA4C is required for the effect of GB on chilling tolerance and that treatment with GB enhances stress tolerance, at least to some extent, via gene activation. A tomato cDNA microarray was used to compare gene expression in flower buds of wild-type and codA-transgenic tomato plants [23]. The expression of 30 genes was 503
Review enhanced and that of 29 genes was repressed in the flower buds of the codA-transgenic plants as compared with wildtype plants. Among the genes whose expression was enhanced, several are known to be involved in cell division, such as the genes for cyclin D (CycD3-1), cyclin-dependent protein kinase p34/cdc2 (CDKA1) and related-to-ubiquitinconjugating enzyme (RCE1). In addition, analysis by semiquantitative reverse transcription (RT)-PCR revealed that GB repressed the expression of the fw2.2 gene, whose product acts as a negative regulator of cell division in the early stages of fruit development [41] in flowers but not in young leaves and flower buds. Taken together, the various observations discussed above indicate that GB, either applied exogenously or synthesized in transgenic plants, seems to be capable of activating specific genes. Identification of GB-inducible genes and the functions of their products will advance our understanding of GB-enhanced stress tolerance in plants. Conclusion and perspectives A variety of genes has been employed to generate transgenic plants that accumulate GB and are tolerant to abiotic stress. Among the properties of these transgenic plants, the physiological and morphological aspects of stress tolerance have been investigated most extensively in codAtransgenic plants. Studies have focused on stress tolerance at all stages of the life cycle of plants, from imbibition of seeds, through the growth of young plants and the photosynthetic activity of mature plants, to the production of fruits and seeds. The protective effects of GB in the reproductive organs of plants have been observed in Arabidopsis, maize and tomato. This protection might have been mediated by the high levels of GB that accumulate in reproductive organs as a consequence of the translocation of GB from other organs, such as leaves. In addition, the codA transgene enhances the productivity of seeds and increases the size of fruits and flowers under non-stress conditions. Further studies should allow the enhancement of stress tolerance in numerous crop species, as well as increases in yield. Possible mechanisms for the GB-enhanced tolerance of plants to various types of abiotic stress include, but are not limited to: (i) protection of the photosynthetic machinery; (ii) induction of specific genes whose products are involved in stress tolerance; (iii) reductions in levels of ROS under stress; and (iv) regulation of the activities of ion-channel proteins either directly or via protection of the plasma membrane. References 1 Rhodes, D. and Hanson, A.D. (1993) Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384 2 Allard, F. et al. (1998) Betaine improves freezing tolerance in wheat. Plant Cell Physiol. 39, 1194–1202 3 Kishitani, S. et al. (1994) Accumulation of glycinebetaine during cold acclimation and freezing tolerance in leaves of winter and spring barley plants. Plant Cell Environ. 17, 89–95 4 Papageorgiou, G.C. and Murata, N. (1995) The unusually strong stabilizing effects of glycinebetaine on the structure and function of the oxygen-evolving photosystem II complex. Photosynth. Res. 44, 243–252
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