Journal of Experimental Botany, Vol. 51, No. 342, pp. 81-88,
January 2000
© 2000 Oxford University Press
Genetic engineering of glycinebetaine synthesis in plants: current status and implications for enhancement of stress tolerance
National Institute for Basic Biology, Okazaki 444–8585, Japan
Received 19 January 1999; Accepted 1 August 1999
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Abstract |
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 Top Abstract IntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Metabolic acclimation via the accumulation of compatible solutes is regarded as a basic strategy for the protection and survival of plants in extreme environments. Certain plants accumulate significant amounts of glycinebetaine (betaine), a compatible quaternary amine, in response to high salinity, cold and drought. It is likely that betaine is involved in the protection of macrocomponents of plant cells, such as protein complexes and membranes, under stress conditions. Genetic engineering of the biosynthesis of betaine from choline has been the focus of considerable attention as a potential strategy for increasing stress tolerance in stress-sensitive plants that are incapable of synthesizing this compatible/protective solute. Three distinct pathways for the synthesis of betaine have been identified in spinach, Escherichia coli and Arthrobacter globiformis, and various genes and cDNAs for the proteins involved are available. Moreover, each of the pathways has been exploited to a greater or lesser extent in efforts to convert betaine-deficient plants to betaine accumulators. In this review, the potential of several recent examples of transgenic approaches to the enhancement of stress tolerance in plants is summarized and discussed.
Key words: compatible solute, genetic engineering, glycinebetaine, stress tolerance, transgenic plants
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Introduction |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Environmental stress severely restricts the distribution and productivity of plants. In particular, salinity and drought are two major constraints that limit agricultural production world-wide (Boyer, 1982
). Plants have evolved various protective mechanisms that allow them to acclimate to unfavourable environments for continued survival and growth. One such mechanism that is ubiquitous in plants is the accumulation of certain organic metabolites of low molecular weight that are known collectively as compatible solutes (Bohnert et al., 1995). Metabolites that serve as compatible solutes differ among plant species and include polyhydroxylated sugar alcohols, amino acids and their derivatives, tertiary sulphonium compounds and quaternary ammonium compounds (Bohnert and Jensen, 1996). There is general agreement that the major role of these metabolites is to serve as organic osmolytes with compatible properties at high concentrations; such osmolytes increase the ability of cells to retain water without disturbing normal cellular functions (Yancey et al., 1982). Genetic engineering to increase levels of some compatible solutes, such as mannitol and proline, appears to be a promising approach in efforts to increase the ability of plants to tolerate environmental stress (for a review, see Hayashi and Murata, 1998, and references therein). In this brief overview, the authors focus on recent progress in the enhancement of stress tolerance in transgenic plants through the genetic engineering of the ability to synthesize glycinebetaine, a compatible solute with strong potential for improving stress tolerance. |
Glycinebetaine |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Glycinebetaine (N,N,N-trimethylglycine; hereafter betaine) is a quaternary ammonium compound that occurs naturally in a wide variety of plants, animals and microorganisms (Rhodes and Hanson, 1993
). It is a dipolar but electrically neutral molecule at physiological pH. With respect to protection against osmotic stress, betaine is regarded as being a particularly effective compatible solute (Le Rudulier et al., 1984). The action of betaine in vivo is not, however, confined to osmoregulation. Numerous experiments in vitro have indicated that betaine acts as an osmoprotectant by stabilizing both the quaternary structure of proteins and the highly ordered structure of membranes against the adverse effects of high salinity and extreme temperatures (for a review, see Gorham, 1995). In photosynthetic systems, for example, betaine efficiently protects various components of the photosynthetic machinery, such as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the oxygen-evolving photosystem II (PSII) complex, from salt-induced inactivation and dissociation into subunits (for a review, see Papageorgiou and Murata, 1995).
The accumulation of betaine in response to salt, drought and cold has been widely recognized in higher plants that are natural accumulators of this compound (Gorham, 1995). Reflecting the beneficial effects of betaine in vitro, a positive correlation exists between the accumulation of betaine and the acquisition of tolerance to salt and cold in maize (Zea mays) and barley (Hordeum vulgaris), respectively (Rhodes et al., 1989; Kishitani et al., 1994). Genetic evidence also indicates that betaine improves the salt tolerance of these members of Gramineae (Grumet and Hanson, 1986; Saneoka et al., 1995). Moreover, exogenous application of betaine to leaves or roots has been shown to increase the tolerance to various stresses of several species of plants, including both natural accumulators and non-accumulators (Itai and Paleg, 1982; Harinasut et al., 1996; Mäkelä et al., 1996; Allard et al., 1998; Hayashi et al., 1998). The fact that many agronomically important crops, such as rice and potato, are betaine-deficient has inevitably led to proposals that it might be possible to increase stress tolerance by genetic manipulation that would allow non-accumulators or low-level accumulators to accumulate betaine at protective levels (McCue and Hanson, 1990).
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Biosynthesis of betaine |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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In most organisms, betaine is synthesized as a result of the two-step oxidation of choline via betaine aldehyde, a toxic intermediate (Fig. 1
). In several higher plants from taxonomically unrelated families, the relevant enzymes are choline monooxygenase (CMO), a ferredoxin-dependent soluble Rieske-type protein, and betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8), a soluble NAD+-dependent enzyme (Weigel et al., 1986; Brouquisse et al., 1989). These enzymes are found mostly in the chloroplast stroma and their activities, as well as levels of betaine, increase in response to salt stress (Hanson et al., 1985). BADH has also been found in several plants that barely accumulate any betaine (Weretilnyk et al., 1989). In mammalian cells and in microorganisms such as Escherichia coli, betaine is synthesized by choline dehydrogenase (CDH; EC 1.1.99.1), a membrane-bound oxygen-dependent enzyme, in combination with BADH (Wilken et al., 1970; Landfald and Strøm, 1986). In contrast to these two pathways that each involve two enzymes, the biosynthesis of betaine is catalysed by a single flavoenzyme, choline oxidase (COD; EC 1.1.3.17), in certain microorganisms, such as the soil bacterium Arthrobacter globiformis (Ikuta et al., 1977; Fig. 1). As far as is known, to date this enzyme has only been found in microorganisms.
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Genetic engineering |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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The enzymes involved in the biosynthesis of betaine whose genes or cDNAs have been cloned to date are from bacteria and/or plants (Weretilnyk and Hanson, 1990
; Lamark et al., 1991; Deshnium et al., 1995; Rathinasabapathi et al., 1997). The introduction, by molecular genetic manipulation, of biosynthetic pathways to betaine has been attempted in non-accumulating species of cyanobacteria (Deshnium et al., 1995; Nomura et al., 1995) and in higher plants (see Table 1 and references therein). The strategy used to generate transgenic plants can be summarized as follows: the relevant gene is introduced under the transcriptional control of a strong promoter that ensures high-level expression of the gene in transgenic plants (e.g. the constitutively active promoter of the gene for 35S rRNA from cauliflower mosaic virus or the light-inducible promoter of a gene for the small subunit of Rubisco). For appropriate localization of enzymes of prokaryotic origin, the gene has often been modified so that the encoded polypeptide is transported post-translationally into the chloroplasts of the engineered plants. The transit peptide of the small subunit of Rubisco has been used as a signal for such transport.
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Introduction of the choline monooxygenase/betaine aldehyde dehydrogenase pathway |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Expression of betaine aldehyde dehydrogenase (BADH) from various plants in tobacco (Nicotiana tabacum)
An initial attempt to introduce the choline mono-oxygenase/betaine aldehyde dehydrogenase pathway in transgenic plants was made by transformation of tobacco with the cDNA for BADH from spinach (Spinacia oleracia) and sugarbeet (Beta vulgaris), two species of typical natural accumulators (Rathinasabapathi et al., 1994
). Expression of each cDNA for BADH was controlled by the 35S promoter and in both cases transgenically produced BADH was correctly targeted to the chloroplasts of the transgenic tobacco. Levels of activity of BADH were similar to or higher than those found in spinach leaves after exposure to salt stress. Expression of foreign BADH conferred resistance to betaine aldehyde since BADH converted this toxic compound to betaine. However, transgenic plants failed to accumulate betaine in the absence of exogenously supplied betaine aldehyde (Table 1), apparently because the enzyme required for the oxidation of choline was not present. Thus, increased expression of BADH alone was insufficient for the synthesis of betaine in the transgenic plants.
A similar result was obtained for tobacco plants transformed with a cDNA for barley BADH (Ishitani et al., 1995). The enzyme from the monocotyledon barley was localized in the peroxisomes of the transgenic plants (Nakamura et al., 1997). This observation stands in marked contrast to the localization in chloroplasts of BADH from chenopods. However, all BADHs from monocotyledons reported to date contain a signal for targeting to microbodies at the carboxyl-terminus so this result is easily explained. Nevertheless, it remains unclear whether microbodies/peroxisomes are the site of betaine synthesis in Gramineae because the choline-oxidizing enzyme has not been identified. Furthermore, it was demonstrated that BADH is not substrate-specific, as had been reported previously, and that BADH might possibly participate in other metabolic pathways, such as the catabolism of polyamines (Trossat et al., 1997).
Expression of choline monooxygenase (CMO) from spinach in tobacco
Transgenic tobacco that constitutively expressed a cDNA for spinach CMO was generated recently and the newly synthesized enzyme was successfully transported to chloroplasts where it was functional (Nuccio et al., 1998). The levels of the foreign CMO in leaves were 10- to 100-fold lower than those in spinach leaves and leaves of the transgenic tobacco plants accumulated betaine at very low levels (no more than 0.05 µmol g-1 FW). The cited authors assumed, from the results of their biochemical characterization of the transgenic plants, that the availability of endogenous choline, which originates from phosphorylcholine and phosphatidylcholine, limited the capacity of the engineered tobacco plants to synthesize betaine. However, it does not seem likely that this assumption has general applicability. For example, tobacco contains only small amounts of choline (0.05 µmol g-1 FW; Nuccio et al., 1998) whereas this compound is present at nearly 30-fold higher levels in Arabidopsis thaliana (Hayashi et al., 1997) and at 10- to 20-fold higher levels in rice (Oryza sativa; Rathinasabapathi et al., 1993; Sakamoto et al., 1998). Moreover, in these experiments, it was found that levels of choline in Arabidopsis and rice were not changed significantly by transformationof plants with a gene for choline oxidase from Arthrobacter (Hayashi et al., 1997; Sakamoto et al., 1998). Furthermore, the level in Arabidopsis plants of phosphatidylcholine was also not affected by such transformation (Alia et al., 1999). Thus, the availability of choline did not seem to limit the synthesis of betaine in these plants.
In the above-mentioned transgenic tobacco that expressed CMO, inefficient synthesis of betaine might have been attributable, at least in part, to the absence of engineered BADH activity in chloroplasts. The endogenous activity of tobacco BADH is relatively low and it is possible that this enzyme is localized in organelles other than chloroplasts (Weretilnyk et al., 1989; Holmstrøm et al. 1994; Rathinasabapathi et al., 1994). By contrast, the activity of BADH in natural accumulators, such as spinach, under stress is high and the enzyme is localized almost exclusively in chloroplasts (Weigel et al., 1986). Therefore, the presence in chloroplasts of both CMO and BADH might be necessary for efficient production of betaine in transgenic plants when attempts are made to introduce the plant pathway into non-accumulators.
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Exploitation of the choline dehydrogenase/betaine aldehyde dehydrogenase pathway from E. coli |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Expression of betaine aldehyde dehydrogenase (BADH) from E. coli in tobacco
Tobacco was transformed with the betB gene for BADH from E. coli that was under the control of a strong light-inducible promoter (Holmstrøm et al., 1994
). Two different gene constructs were used for transformation in order to target the encoded protein either to the chloroplasts or to the cytosol of transgenic plants (Table 1). This work yielded essentially the same results as those obtained after transformations with cDNAs for plant BADHs, indicating once again the requirement for introduction of a second enzyme, namely a choline-oxidizing enzyme.
Expression of choline dehydrogenase (CDH) from E. coli in tobacco
Choline dehydrogenase of E. coli catalyses the first step in the synthesis of betaine, the oxidation of choline. However, this enzyme also catalyses the second step, the dehydrogenation of betaine aldehyde that yields betaine (Landfald and Strøm, 1986). Tobacco was transformed with the betA gene for CDH under the control of a constitutively expressed promoter (Lilius et al., 1996). Since the protein from E. coli lacks a targeting signal, the transgenically expressed protein was presumably associated with the plasma membrane, and was not imported into subcellular organelles. Expression of CDH in tobacco increased the total CDH activity 4.5- to 6-fold. However, no accumulation of betaine was demonstrated in the transgenic plants (Table 1). Therefore, although the cited authors reported that transformation of tobacco with the gene for CDH enhanced tolerance to salt at levels of NaCl as high as 300 mM in terms of growth yield and visible injury to leaves, the enhanced tolerance might not have been related to the accumulation of betaine.
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Genetic engineering using the gene for choline oxidase from Arthrobacter |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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Expression of choline oxidase (COD) from Arthrobacter in Arabidopsis
The major advantage of using COD, as distinct from CMO/BADH or CDH/BADH, as a tool for engineering the synthesis of betaine is that the introduction of only a single gene (codA) for this enzyme is sufficient for the conversion of choline to betaine in transgenic plants (Fig. 1). Arabidopsis thaliana was transformed with a modified codA gene that encoded COD with a signal for targeting to chloroplasts (Hayashi et al., 1997
). The transformation resulted in the accumulation of betaine in leaves at 1.0 µmol g-1 FW while wild-type plants contained no detectable betaine. On the assumption that betaine is localized exclusively in the chloroplasts, its concentration was estimated to be about 50 mM (Hayashi et al., 1997). Seeds of transgenic plants also accumulated betaine at 12–18 µmol g-1 DW (Hayashi et al., 1998). These experiments provided the first example of genetically engineered plants with the ability to synthesize betaine at appreciable levels.
In order to examine the role of betaine in vivo, members of this group studied the responses of transgenic Arabidopsis that expressed COD to various stresses at various stages of development (Hayashi et al., 1997, 1998; Alia et al., 1998a, b, 1999; Table 1). Transgenic strains were more tolerant to salt stress than the wild-type strains during germination of seeds (up to 300 mM NaCl) and during the early and late stages of development of plants (up to 200 mM NaCl; Fig. 2; Hayashi et al., 1997, 1998). In addition to enhanced tolerance to salt stress, transgenic Arabidopsis exhibited significantly enhanced tolerance to both low- and high-temperature stress (Hayashi et al., 1997; Alia et al., 1998a, b). Seeds of transgenic plants remained able to germinate after the exposure to extreme temperatures, such as 0 °C and 50 °C, during imbibition (Fig. 3). Germination and the subsequent growth of seedlings of transgenic plants were more rapid than those of wild-type Arabidopsis at both low and high temperatures (Alia et al., 1998a, b). Transgenic plants were also visibly less damaged than wild-type plants after exposure to low temperature in the light (Hayashi et al., 1997). In these physiological evaluations of stress tolerance, the degree of tolerance was correlated in every case with the level of betaine in transgenic Arabidopsis.
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Enhanced tolerance was also evident at the cellular level in transgenic Arabidopsis that expressed COD. An analysis of the fluorescence of chlorophyll a revealed that the activity of PSII was maintained at a higher level in transgenic plants than in wild-type plants under salt stress, cold stress and high-intensity light stress (Hayashi et al., 1997
; Alia et al., 1999). Enhanced tolerance of the photosynthetic machinery to high-intensity light was the result of accelerated recovery of the PSII complex from photo-induced inactivation (Alia et al., 1999). This accelerated recovery involved the synthesis of proteins de novo and was not accompanied by changes in the levels of unsaturation of membrane lipids (Alia et al., 1999), which has been shown to be an important mechanism for maintenance of the photosynthetic machinery under strong light (Nishida and Murata, 1996).
The possible side effects of the introduction of the gene for COD were examined since the enzyme produces hydrogen peroxide as a by-product of catalysis (Alia et al., 1999). Leaves of transgenic Arabidopsis that expressed COD did have elevated levels of hydrogen peroxide, but increases were only 50% to 100% of levels in wild-type plants under stress and non-stress conditions (Alia et al., 1999). Moreover, the activities of enzymes that are responsible for scavenging hydrogen peroxide, namely, ascorbate peroxidase and, to a lesser extent, catalase, were significantly higher in transgenic plants than in wild-type plants (Alia et al., 1999). These observations suggest that the hydrogen peroxide generated by choline oxidase might have stimulated the expression of scavenging enzymes with the resultant maintenance of intracellular levels of hydrogen peroxide within a certain limited range.
To summarize, the accumulation of betaine in transgenic Arabidopsis as a result of expression of COD from Arthrobacter enhanced tolerance to salt, cold, heat, and high-intensity light stress (Table 1). Enhancement of tolerance was not confined to specific stages of development but was recognized throughout the course of various developmental stages that included the imbibition and germination of seeds, as well as the early and later stages of vegetative growth. In addition, recent studies have revealed that the transgenic plants had improved tolerance to cold during reproduction and also developed tolerance to freezing at maturity (N Murata, unpublished data).
Expression of choline oxidase (COD) from Arthrobacter in rice
Transgenic rice plants were generated similarly to the transgenic Arabidopsis plants described above with COD targeted either to the chloroplasts or to the cytosol. In the former case, betaine accumulated at about 1 µmol g-1 FW in leaves; in the latter plants, it accumulated at about 5 µmol g-1 FW (Sakamoto et al., 1998). Transgenic rice plants with COD targeted either to chloroplasts or to the cytosol exhibited enhanced tolerance to salt- or cold-induced photoinhibition. Moreover, the photosynthetic machinery was more efficiently protected when COD was targeted to the chloroplasts than to the cytosol of transgenic rice (Sakamoto et al., 1998). This observation indicates that the subcellular site of betaine synthesis might be important in efforts to improve the stress tolerance of plants.
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Possible mechanisms of stress tolerance |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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While genetic engineering has allowed engineered plants to produce betaine, there are considerable differences in levels of betaine, on a fresh weight basis, between transgenic plants (0.05–5 µmol g-1 FW) and natural accumulators under stress conditions (4–40 µmol g-1 FW; Rhodes and Hanson, 1993
). The amounts of betaine accumulated suggest that the enhancement of stress tolerance can hardly be attributed to any action of betaine that involves osmotic adjustment to the external environment. A major role of betaine might be to protect membranes and macromolecules from the damaging effects of stress. It is also possible that betaine might be compartmentalized within cells such that, at certain sites, the concentration of betaine might be high enough to confer substantial protection against stress even when the overall level of accumulation is low. Such a possibility is supported by the observations that betaine is concentrated exclusively in the cytoplasm, and not in the vacuoles, of leaves of salt-grown halophytes (Matoh et al., 1987), and levels of betaine as low as 5 µmol g-1 FW can protect some natural accumulators from the damaging effects of stress (Arakawa et al., 1990; Ishitani et al., 1993). Moreover, recent studies have provided new insights into the possible roles of betaine in cells under stress. Betaine destabilizes double-helixed DNA and lowers the melting temperature of DNA in vitro. Thus, betaine might play a role in promoting transcription and replication under high-salt conditions (Rajendrakumar et al., 1997). The physiological characterization of transgenic plants (Alia et al., 1999) suggested that betaine might accelerate protein synthesis de novo during recovery from stress. Such possibilities require further detailed examination in future studies. |
Conclusions |
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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The following conclusions related to enhancement of the stress tolerance of plants can be drawn from experiments with genetically modified plants. (1) In order to establish a pathway for the appropriate synthesis of betaine, the oxidation of choline is a prerequisite for genetic manipulation in many cases. (2) For the engineering of plants that will synthesize betaine, microorganisms appear to be a useful source of genes, as exemplified by the codA gene from Arthrobacter and the betA gene from E. coli. (3) The synthesis of betaine in chloroplasts can enhance tolerance to several types of stress. (4) The synthesis of betaine in chloroplasts can be associated with enhanced tolerance at various stages of development. It is anticipated that, in the future, the results from the basic research summarized in this review will be fully exploited for the improvement of stress tolerance in agronomically valuable crops. One of the next necessary steps will be to increase still further the levels of accumulated betaine without associated negative effects on engineered plants. Choline is synthesized in the cytosol and, therefore, it must be transported into chloroplasts by an as yet unidentified transporter on the chloroplast envelope. Genetic engineering that results in efficient transport or uptake of choline into chloroplasts is another possible avenue for future research that might lead to enhanced tolerance of plants to environmental stress.
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Acknowledgments |
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This work was supported, in part, by a Grant-in-Aid for Specially Promoted Research (no. 08102011) from the Ministry of Education, Science and Culture, Japan. It was also supported by the National Institute for Basic Biology Cooperative Research Program for Molecular Mechanisms of Stress Responses.
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Notes |
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1 To whom correspondence should be addressed. Fax: +81 564 54 4866. E-mail: murata@nibb.ac.jp
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Abbreviations |
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BADH, betaine aldehyde dehydrogenase; betaine, glycinebetaine; CDH, choline dehydrogenase; CMO, choline monooxygenase; COD, choline oxidase; PSII, photosystem II; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase.
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TopAbstractIntroductionGlycinebetaineBiosynthesis of betaineGenetic engineeringIntroduction of the choline...Exploitation of the choline...Genetic engineering using the...Possible mechanisms of stress...ConclusionsReferences |
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