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Keywords:

  • abiotic stress tolerance;
  • antioxidative defence;
  • glycinebetaine;
  • photoinhibition;
  • reactive oxygen species

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Various compatible solutes enable plants to tolerate abiotic stress, and glycinebetaine (GB) is one of the most-studied among such solutes. Early research on GB focused on the maintenance of cellular osmotic potential in plant cells. Subsequent genetically engineered synthesis of GB-biosynthetic enzymes and studies of transgenic plants demonstrated that accumulation of GB increases tolerance of plants to various abiotic stresses at all stages of their life cycle. Such GB-accumulating plants exhibit various advantageous traits, such as enlarged fruits and flowers and/or increased seed number under non-stress conditions. However, levels of GB in transgenic GB-accumulating plants are relatively low being, generally, in the millimolar range. Nonetheless, these low levels of GB confer considerable tolerance to various stresses, without necessarily contributing significantly to cellular osmotic potential. Moreover, low levels of GB, applied exogenously or generated by transgenes for GB biosynthesis, can induce the expression of certain stress-responsive genes, including those for enzymes that scavenge reactive oxygen species. Thus, transgenic approaches that increase tolerance to abiotic stress have enhanced our understanding of mechanisms that protect plants against such stress.


Abbreviations
3-PGA

glycerol-3-phosphate

Cyt b6f

cytochrome b6f complex

DHAP

dihydroxyacetone phosphate

e-

electron

GB

glycinebetaine

LHC

light-harvesting complex

pre-D1 protein

precursor to D1 protein

RuBP

ribulose-1,5-bisphosphate

INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

When plants are exposed to stressful conditions, metabolic shifts occur and result in changes in the levels of a variety of cellular metabolites. Such modifications in response to abiotic stress appear to be associated with the enhanced ability to tolerate such conditions. Metabolites that might be expected to contribute to enhanced stress tolerance include soluble sugars, amino acids, organic acids, polyamines, and lipids (Guy 1990). One important group of such metabolites includes the so-called ‘compatible solutes’, which are small organic metabolites that are very soluble in water and are non-toxic at high concentrations. One of the best-studied compatible solutes is glycinebetaine (N,N,N-trimethylglycine, abbreviated as GB; Chen & Murata 2002; Chen & Murata 2008).

GB is a quaternary ammonium compound that is found in bacteria, haemophilic archaebacteria, marine invertebrates, plants and mammals (Rhodes & Hanson 1993; Chen & Murata 2002; Takabe, Rai & Hibino 2006; Chen & Murata 2008). It accumulates to osmotically significant levels in many salt-tolerant plants (Rhodes & Hanson 1993) and halotolerant cyanobacteria (Chen & Murata 2008). Levels of GB vary considerably among plant species and organs, as shown in Table 1 (see ‘non-stressed levels’). Plants in many taxonomically distant species normally contain low levels of GB (these plants are known as natural accumulators of GB), but they accumulate larger amounts of GB when subjected to abiotic stress (Storey, Ahmad & Wyn Jones 1977). In many other species, no GB is detectable under normal or stressful conditions. There is now strong evidence that GB plays an important role in tolerance to abiotic stress.

Table 1.  Examples of the stress-induced accumulation of GB in plants that naturally accumulate GB
Plant speciesGB contentOrganStress treatmentReference
Non-stressedStressed
µmol g−1 FWµmol g−1 FW
  1. DW, dry weight; FW, fresh weight.

Amaranthus tricolor2∼10Leaves0.3 m NaClBhuiyan et al. 2007
Atriplex halimus∼1055Shoots15% PEG 10 000Hassine et al. 2008
∼1043Shoots0.16 m NaClHassine et al. 2008
Atriplex spongiosa1545Leaves0.8 m NaClStorey & Wyn Jones 1979
Gossypium hirsutum L.3.56.7Leaves0.15 m NaClDesingh & Kanagaraj 2007
Hordeum vulgare L.1.55.56th leaf0.2 m NaClHattori et al. 2009
0.11.0Roots0.2 m NaClHattori et al. 2009
Sorghum bicolor L.49Leaves0.1 m NaClYang et al. 2003
Spinacia oleracea525Leaves0.3 m NaClMcCue & Hanson 1990
0.0211Leaves0.17 m NaClMaritino et al. 2003
Suaeda monoica5085Shoots1 m NaClStorey & Wyn Jones 1979
Triticum aestivum L.     
 cv. Glenlea6.515.3LeavesCold (6/2°C)Allard et al. 1998
 cv. Fredrick8.521.3LeavesCold (6/2°C)Allard et al. 1998
Zea mays1.22.0Leaves10% PEG 6000Quan et al. 2004b
1.02.9Leaves0.15 m NaClRhodes et al. 1989
µmol g−1 DWµmol g−1 DW   
Atriplex spongiosa150340Leaves0.5 m NaClStorey et al. 1977
Avicennia marina67∼150Leaves0.4 m NaClHibino et al. 2001
12∼25Roots0.4 m NaClHibino et al. 2001
Beta maritima91–103245–258Shoots0.15 m NaClHanson & Wyse 1982
Fragaria × ananassa Duch.1.83.7Leaves4-week coldRajashekar et al. 1999
Hordeum vulgare L.20–3148–65ShootsWater stressLadyman et al. 1983
Spartina × townsendii160320Shoots0.3 m NaClStorey et al. 1977
Suaeda monoica280580Shoots0.5 m NaClStorey et al. 1977
Triticum aestivum L.∼70∼105Leaves30% PEG-6000Wang et al. 2010

The biological functions of GB have been studied extensively in higher plants, such as spinach, sugar beet, barley and maize (Rhodes & Hanson 1993; Chen & Murata 2008). The availability of GB-accumulating transgenic plants has provided insights into the way in which GB protects plants cells. Furthermore, many lines of GB-accumulating transgenic plants exhibit greatly improved tolerance to various types of abiotic stress and their properties suggest promising strategies for the development of stress-tolerant crop plants.

In this review, we summarize and discuss current understanding of the biosynthesis of GB and the mechanisms whereby it increases the ability of plants to tolerate abiotic stress. Several reviews of the relationship between GB and tolerance to abiotic stress have appeared recently (Sakamoto & Murata 2000; Chen & Murata 2002, 2008; Takabe et al. 2006), but progress in the field has been rapid and a fresh appraisal is appropriate. Research on transgenic plants that accumulate compatible solutes other than GB has shown that they also exhibit increased tolerance to various types of abiotic stress, but we shall not discuss these plants here. The interested reader is referred to our earlier review (Sakamoto & Murata 2000; Chen & Murata 2002) and to recent papers, such as those by Bhatnagar-Mathur, Vadez & Sharma (2008), Valliyodan & Nguyen (2006), Miller et al. (2010), and Ashraf (2009).

BIOSYNTHESIS AND ACCUMULATION OF GB

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Pathways of GB biosynthesis

Two biosynthetic pathways lead to the generation of GB (Chen & Murata 2002). In the majority of biological systems, including most animals, plants and microorganisms, GB is synthesized from choline by a two-step oxidation reaction: choline is oxidized to betaine aldehyde, which is oxidized to yield GB. The first oxidation is catalysed by choline monooxygenase (CMO) in plants and by choline dehydrogenase (CDH) in animals and bacteria (encoded by the betA gene), and the second oxidation is catalysed by NAD+-dependent betaine aldehyde dehydrogenase (BADH) (Chen & Murata 2002; Takabe et al. 2006). In the soil bacterium Arthrobacter sp., GB is synthesized from choline by a single enzyme, choline oxidase, which is encoded by the codA gene (Chen & Murata 2002).

In GB-deficient plants, such as tobacco, the absence of CMO is a primary constraint on GB synthesis whereas the endogenous supply of choline is also problematic (Nuccio et al. 1998). Transgenic tobacco plants that constitutively express cDNA for spinach CMO produced a low level of GB, namely, 0.05 µmol g−1 fresh weight (Nuccio et al. 1998). When CMO+ plants were supplied with 5 mm choline or phosphocholine, the GB level increased by at least 30-fold.

In plants, the key enzyme of choline synthesis is phosphoethanolamine: N-methyltransferase (PEAMT; EC 2.1.1.103), a cytosolic enzyme, which catalyses all three steps of methylation reactions in the conversion of phospohoethanolamine to phosphocholine (McNeil et al. 2001). Overexpression of spinach PEAMT in transgenic tobacco plants increased the level of phosphocholine by 5-fold and that of choline by 50-fold, resulting in a 30-fold increase in the level of GB via an engineered pathway of GB synthesis (McNeil et al. 2001).

Peel, Mickelbart & Rhodes (2010) utilized near-isogenic lines for GB accumulation to characterize the biochemical basis for GB deficiency in maize and sorghum. In the presence of NaCl, GB-non-accumulating lines had increased concentrations of choline and phosphocholine, but not GB. Surprisingly, the lack of GB accumulation in GB-non-accumulating lines was found not due to the lack of the CMO gene or CMO protein. The decreased synthesis of GB can be explained by the elevated concentration of endogenous phosphocholine, which is a strong inhibitor of PEAMT in vitro.

The second pathway has only been found in the extreme-halophytic phototrophic bacteria Actinopolyspora halophila and Ectothiorhodospira halochloris. In these bacteria, GB is synthesized from glycine by a three-step N-methylation reaction (Chen & Murata 2002; Takabe et al. 2006). Glycine is methylated to yield, firstly, sarcosine and then dimethylglycine, and, in the final methylation step, GB is formed. S-Adenosylmethionine is the donor of each of the methyl groups. The three reactions of methylation are catalysed by two enzymes, namely, glycinesarcosine methyltransferase (GSMT) and sarcosine dimethylglycine methyltransferase (SDMT). The first reaction is catalysed by GSMT and the third is catalysed by SDMT. The second step is catalysed by both GSMT and SDMT. The two enzymes have partially overlapping substrate-specificity: both glycine and sarcosine serve as substrates for GSMT, while both sarcosine and N,N-dimethylglycine serve as the substrates for SDMT.

Localization of GB in plant cells

There is no clear evidence for the compartmentalized localization of GB at specific sites in plant cells. In spinach plants, the amount of GB in the chloroplasts is estimated to be close to 50% of the total amount of GB in leaves (Robinson & Jones 1986). The cellular localization of the remaining 50% of the GB in leaves remains to be clarified. It is reasonable to assume that the remaining GB is located in the cytoplasm to provide osmotic balance. Since the vacuole occupies approximately 90% of each cell's volume, the possible presence of GB in vacuoles has been examined. In Suaeda maritima, GB was found to have been excluded from the vacuoles in freeze-substituted leaf cells (Hall, Harvey & Flowers 1978). By contrast, in vacuoles isolated from the storage-root tissue of red beet (Beta vulgaris L.), the vacuolar pool accounted for 26 to 84% of the total GB in the tissue (Leigh, Ahmad & Wyn Jones 1981). Since these results were obtained close to three decades ago, the compartmentalization of GB in plant cells should be re-examined by new and relevant methods for the separation of organelles and the quantitation of GB.

Sites of GB biosynthesis in higher plants

Enzymes involved in the synthesis of GB have been studied in members of Chenopodiaceae, such as spinach and sugar beet (Hanson et al. 1985; Weigel, Weretilnyk & Hanson 1986; Weretilnyk & Hanson 1990; Rathinasabapathi et al. 1997); in Amaranthaceae (Ling et al. 2001; Bhuiyan et al. 2007), in particular, Atriplex nummularia (Tabuchi et al. 2005); and in members of Gramineae, such as barley (Nakamura et al. 2001; Fujiwara et al. 2008). Although both CMO and BADH are localized in chloroplasts, they are encoded by nuclear genes that include transit sequences for targeting to chloroplasts. This scenario might seem to indicate that GB is synthesized only in chloroplasts. However, in some naturally GB-accumulating plants, such as mangrove [Avecennia marina (Forsk.)] (Hibino et al. 2001) and barley (Fujiwara et al. 2008), no CMO activity was detected in chloroplasts. It is possible that, in mangrove and barley, some enzyme that is not specific to GB synthesis but has CMO activity might catalyse the first step in the oxidation of choline. The nature and cellular location of such enzyme(s) in the cell are unknown. Moreover, the conversion of choline to betaine aldehyde might not have an absolute requirement for CMO. In several cases, transformation of non-GB accumulators with a single gene for BADH was sufficient to bring about the accumulation of GB. Such non-GB-accumulating plants might have low levels of CMO activity or they might have some other enzyme(s) that can catalyse the first step in the oxidation of choline. Moreover, BADH might not function exclusively in the conversion of betaine aldehyde to GB. For example, BADH catalyses the oxidation and/or detoxification of a variety of compounds, which include dimethylsulfoniopropionaldehyde and aminoaldehydes (Trossat, Rathinasabapathi & Hanson 1997).

There are two genes for BADH in the genome of barley: BBD1 and BBD2 (Nakamura et al. 2001; Fujiwara et al. 2008). BBD1 encodes a peroxisomal enzyme, while BBD2 encodes a cytosolic enzyme (Fujiwara et al. 2008). Although this finding does not exclude the possibility that BADH or its activity might be present in chloroplasts, it challenges the conclusion, reached as a result of research in Chenopodiaceae, that both steps in the synthesis of GB occur exclusively in the chloroplast. If GB can be synthesized by cytosolic BBD2 in barley, the first step in the oxidation of choline might also occur in the cytosol. The same might hold true for BBD1 in the peroxisome. Further research is needed to confirm the biosynthesis of GB in the cytosol.

In barley plants, the accumulation of GB increases under salt stress and the extent of such accumulation is greater in young leaves than in old ones (Fujiwara et al. 2008). The level of BBD2 transcripts rises considerably in the vascular parenchyma cells of salt-stressed plants. In roots under salt stress, BBD1 transcripts are detected in epidermal cells, while BBD2 transcripts are detected in the pericycle. BADH itself is detected around the xylem vessels of leaves, and in the pericycle and epidermal cells of roots of salt-stressed plants. These results indicate that GB is synthesized in the vascular tissues of leaves and in the pericycle of roots.

Transporters of GB

Exogenously applied GB is readily taken up by plant cells, but little is known about the transport of GB in plant cells. It is likely that transporters of GB are located in the plasma membrane, but no GB-specific transporter has been reported to date. The transport of GB from the cytosol to various subcellular compartments is also poorly understood. In spinach, for example, the majority of GB that accumulates in response to salt stress is found in the chloroplasts, and the concentrations of GB in chloroplasts isolated from control and salt-stressed spinach plants were 0.7 and 6.6 µmol mg−1 chlorophyll, respectively (Robinson & Jones 1986). The resultant concentration gradient across the chloroplast envelope suggests the existence of a specific transport mechanism. Although little is known about the transport of compatible solutes in plant cells, Schwacke et al. (1999) demonstrated that the product of the tomato gene LeProT1, a homolog of a proline transporter in Arabidopsis, transported GB with high affinity and both proline and γ-amino butyric acid (GABA) with low affinity, when expressed in yeast. Similar results were also reported for a GABA transporter, ProT2, whose gene was cloned from Arabidopsis (Breitkreuz et al. 1999). In this case, the ProT2-mediated transport of GABA was strongly inhibited by GB, indicating that GB had strong affinity for the transporter. These results suggest that transporters both of proline and of GABA might function in the transport of GB. Ueda et al. (2001) cloned the gene for a proline transporter (HvProT) from the roots of salt-stressed barley. They demonstrated that the uptake of proline by the yeast cells that expressed HvProT was not inhibited by GB, suggesting that HvProT does not act as a transporter of GB.

Translocation of GB in plants

The translocation of 14C-labeled GB in barley plants demonstrated that GB is translocated, most probably, via the phloem (Ladyman, Hitz & Hanson 1980). Heat girdling of the leaf sheath prevented the export of [14C]-GB from leaf blades. It appeared that GB, synthesized by mature leaves during exposure of plants to abiotic stress, behaved as an inert end-product, which, upon re-watering of plants, was translocated to the expanding leaves. Mäkeläet al. (1996) reached a similar conclusion from the results of experiments with tomato plants. In sugar beet (B. vulgaris) plants, d11-betaine applied exogenously to old leaves was translocated preferentially into young leaves and roots (Yamada et al. 2009).

When GB was applied to single mature leaves of tomato plants, a large fraction of the incorporated GB was translocated to meristem-containing tissues, which included flower buds and shoot apices (Park & Chen 2006). The varying levels of GB in the different plant organs indicated active and, possibly, regulated translocation from the original site of application and accumulation. Translocation of GB with photosynthetic assimilates has also been reported, in particular to actively growing and expanding parts of plants, indicating that the long-distance transport of GB is phloem-related (Mäkeläet al. 1996). In GB-accumulating transgenic Arabidopsis (Sulpice et al. 2003) and tomato (Park et al. 2004, 2007a) plants, the highest levels of GB are found in actively growing tissues, such as flowers and shoot apices, indicating that GB is efficiently translocated from source to sink tissues via the phloem.

Inducers of the biosynthesis of GB

In plants that accumulate GB naturally, the accumulation of elevated levels of GB is induced by abiotic stress, such as high salt, drought and cold (Jagendorf & Takabe 2001). Abiotic stress is, moreover, not the only inducer of the accumulation of GB. A wide range of compounds has been shown to induce the accumulation of GB in barley plants (Jagendorf & Takabe 2001), including inorganic salts (KCl, MaCl2, LiCl and Na2SO4), oxidants (H2O2 and cumene hydroperoxide) and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid and acetylsalicylic acid). It remains to be determined whether such inducers are also active in other plants that accumulate GB. Further studies with these inducers of the accumulation of GB might provide clues to the signal transduction pathways that induce the accumulation of GB.

GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

The cloning of various genes for enzymes that catalyse the biosynthesis of GB has been reported, and many lines of transgenic plants have been produced that express GB-biosynthetic genes from bacteria (Table 2) and plants (Table 3). The various transgenic plants accumulate GB at a variety of levels and exhibit enhanced tolerance to various types of stress (Tables 2 & 3). Most lines of plants that have been engineered to synthesize GB are derived from plants that are natural non-accumulators of GB and the transgenic plants accumulate only low levels of GB. Within this group of plants, the greatest accumulation of GB was found in codA-transgenic rice plants (5.3 µmol g−1 FW; Sakamoto, Alia & Murata 1998). In a natural accumulator of GB, maize, the highest level of GB accumulated in betA-transgenic plants was 5.7 µmol g−1 FW in leaves (Quan et al. 2004a). This level was higher than that in wild-type (WT) plants subjected to drought stress, indicating that transgenic plants were able to accumulate larger amounts of GB than the maximum amount in WT plants (Table 1).

Table 2.  Transgenic plants that overexpressed bacterial genes for the synthesis of GB and their enhanced tolerance to abiotic stress
Plant speciesGeneSubcellular locationMaximum accumulation (in leaves)Enhanced tolerance to:Reference
  1. N.D., not determined.

Arabidopsis thalianacodAChloroplasts1.2 µmol g−1 FWChilling, saltHayashi et al. 1998
codAChloroplasts1.2 µmol g−1 FWChillingAlia et al. 1998a
codAChloroplasts1.2 µmol g−1 FWHeatAlia et al. 1998b
codAChloroplasts1.2 µmol g−1 FWSaltHayashi et al. 1997
codAChloroplasts1.2 µmol g−1 FWStrong lightAlia et al. 1999
codAChloroplasts1.2 µmol g−1 FWFreezingSakamoto et al. 2000
coxCytosol1.9 µmol g−1 DWSalt, drought, freezingHuang et al. 2000
codAChloroplasts1.2 µmol g−1 FWSaltSulpice et al. 2003
ApGSMT+ApDMT[+5 mm glycine]Cytosol∼2.0 µmol g−1 FWSalt, chilling, CuSO4Waditee et al. 2005
Brassica napuscoxCytosol13 µmol g−1 DWDrought, saltHuang et al. 2000
Brassica junceacodAChloroplasts0.82 µmol g−1 FWSaltPrasad et al. 2000
Diospyras kakicodAChloroplasts0.3 µmol g−1 FWSaltGao et al. 2000
Gossypium hirsutumbetACytosol354 µmol g−1 DWDroughtLv et al. 2007
Lycopersicon esculentumcodAChloroplasts0.3 µmol g−1 FW 1.2 µmol g−1 FW (flowers)Chilling, saltPark et al. 2004
codAChloroplasts23 nmol mg−1 chlorophyllChilling, salt, oxidative stressPark et al. 2007a
codACytosol95 nmol mg−1 chlorophyll  
codAChloroplasts + cytosol120 nmol mg−1 chlorophyll 
Nicotiana tabacumcoxCytosol13 µmol g−1 DWSaltHuang et al. 2000
betACytosol0.035 µmol g−1 FWSalt, chillingHolmstrom et al. 2000
Oryza sativacodAChloroplastsN.D.FreezingKonstantinova et al. 2002
codACytosol5.3 µmol g−1 FWSalt, chillingSakamoto et al. 1998
codAChloroplasts1.1 µmol g−1 FWSalt, chillingSakamoto et al. 1998
betA (modified)Mitochondria5.1 µmol g−1 FWSalt, droughtTakabe et al. 1998
codAChloroplasts2.1 µmol g−1 DWSaltMohanty et al. 2002
coxChloroplasts3.1 µmol g−1 DWSaltSu et al. 2006
Solanum tuberosumcodAChloroplasts1.4 µmol g−1 FWSalt, drought, oxidative stressAhmad et al. 2008
Zea maysbetACytosol5.7 µmol g−1 FWChillingQuan et al. 2004a
betACytosol5.7 µmol g−1 FWDroughtQuan et al. 2004b
Table 3.  Transgenic plants that overexpressed plant genes for the synthesis of GB and their enhanced tolerance to abiotic stress
Plant speciesGene (origin)aSubcellular locationMaximum accumulation (in leaves)Enhanced tolerance to:Reference
  • a

    Plant species from which the gene was cloned.

  • b

    When supplied with 10 mM betaine aldehyde.

  • c

    28/13 °C for 5 weeks.

  • N.D., not determined.

Arabidopsis thalianaCMO + BADH (spinach)N.D.∼0.9 µmol g−1 FWSaltHibino et al. 2002
Nicotiana tabacumBADH (spinach)Chloroplasts4.6 µmol g−1 FWHeatYang et al. 2005, 2007
BADH (Atriplex)N.D.∼7 µmol g−1 DWSaltZhou et al. 2008
CMO (sugar beet)Plastids∼0.25 µmol g−1 FWSalt, droughtZhang et al. 2008
Daucus carotaBADH (spinach)Plastids101 µmol g−1 DWSaltKumar et al. 2004
Lycopersicon esculentumBADH (Atriplex)N.D.0.45 µmol g−1 DWSaltZhou et al. 2007
Oryza sativaBADH (barley) (+betaine aldehyde)bPeroxisome56.4 µmol g−1 DWSalt, cold, heatKishitani et al. 2000
CMO (spinach)Chloroplasts0.45 µmol g−1 DWSalt, temperaturecShirasawa et al. 2006
Triticum aestivum L.BADH (Atriplex)N.D.∼100 µmol g−1 DWDrought, heatWang et al. 2010

Waditee et al. (2005) transformed Arabidopsis with genes for GSMT and SDMT from Aphanothece halophytica (designated ApGSMT and ApSDMT, respectively). They reported that the amounts of GB accumulated were higher than those in plants transformed with genes for choline-oxidizing enzymes. However, the maximum levels of GB in their transgenic plants were close to 2.0 µmol g−1 FW, when plants were supplied with 0.1 m NaCl and 5 mm glycine. Thus, the combined actions of ApGSMT and ApDMT do not appear to be more useful than those of choline-oxidizing enzymes for engineering the biosynthesis of GB in plants.

Su et al. (2006) generated several lines of GB-producing transgenic rice plants, in which the cox gene for choline oxidase from Arthrobacter pascens, fused to a chloroplast-targeting sequence, was expressed under the control of a stress-inducible promoter (SIP) or the promoter of a gene for ubiquitin (UBI), which was constitutively active. The highest level of accumulation of GB (2.6 µmol g−1 DW) in SIP lines that had been grown under saline conditions was not as high as that in UBI lines (3.1 µmol g−1 DW). Therefore, the use of SIP was no more effective for production of GB than the constitutively active UBI promoter. However, saline growth conditions enhanced the accumulation of GB by as much as 89% in the SIP lines, whereas a maximum increase of only 44% was seen in UBI lines. In spite of lower concentrations of GB, the stress tolerance of the SIP lines was significantly higher than that of the UBI lines, suggesting that the stress tolerance of the SIP plants was not attributable solely to increases in GB content.

Early studies of natural accumulators of GB focused on the role of GB in maintaining the osmotic potential of cells, and the effective concentration of GB was assumed to be very high. However, the contribution of stress-induced GB and transgenetically accumulated GB to the total osmotic potential of cells is small and does not fully explain the associated increases in stress tolerance. In all of the previously mentioned lines of transgenic plants, levels of GB were below the range reported for plants that produce GB naturally (Table 1).

With respect to possible advantages of the modification of chloroplast genomes, as distinct from nuclear genomes, in applications to agriculture, there are two reports of the genetic engineering of plastid genomes for GB synthesis. Genetic engineering of the chloroplast genome of carrot, which resulted in the expression of a gene for BADH from spinach, conferred strong tolerance to salt stress (Kumar, Dhingra & Daniell 2004). The highest level of GB was close to 100 µmol g−1 DW, which is the highest level reported in GB-accumulating transgenic plants to date. Zhang et al. (2008) described the genetic transformation of the plastid genome of tobacco plant with the gene for CMO from sugar beet. Levels of GB in the leaves of the resultant tobacco plants ranged from 0.2 to 0.5 µmol g−1 FW, and GB appeared to be localized exclusively in chloroplasts.

Although considerable efforts have been made to increase overall levels of GB in transgenic plants, reported levels of GB are still relatively low when compared to levels in natural accumulators of GB after their exposure to abiotic stress (Table 1). Moreover, we do not yet understand why it has not been possible to achieve higher levels of GB in transgenic plants.

There are two known factors that can limit the accumulation of GB in chloroplasts of transgenic plants: the availability of endogenous choline (Huang et al. 2000) and the transport of choline across the chloroplast envelope (McNeil et al. 2000). Huang et al. (2000) introduced the metabolic steps for oxidation of choline to GB into three diverse species –Arabidopsis thaliana, Brassica napus and tobacco (Nicotiana tabacum). In these species, exogenous supply of choline significantly increased the level of GB accumulation, suggesting that choline supplement is required for the enhancement of GB levels in transgenic plants. Furthermore, the establishment of a model for the labelling kinetics of choline metabolites has revealed that the import of choline into chloroplasts limits GB synthesis in these compartments (McNeil et al. 2000). Furthermore, Nuccio et al. (1998) found that the activity of PEAMT was 30 to 100 times lower in tobacco compared with spinach, suggesting that it may be the main reason why there is a limitation in the endogenous choline supply in tobacco plants, non-GB-accumulators. In plants, the biosynthesis of choline occurs exclusively in the cytosol (McNeil et al. 2000). Their research has provided the first example of engineered biosynthesis of choline, and has demonstrated that GB accumulation can be significantly increased in transgenic plants that normally accumulate GB only to very limited levels (McNeil et al. 2001). Therefore, it is likely that increased activity of PEAMT in non-GB-accumulators may further increase the level of GB accumulation in the transgenic plants.

GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Abiotic stresses inhibit the repair of photosystem II

Exposure of photosynthetic organisms to strong light results in the inactivation of photosystem II (PSII) (Aro, Virgin & Andersson 1993). This phenomenon is referred to as photoinhibition. Since light energy is the driving force for photosynthesis, photoinhibition is unavoidable in photosynthetic organisms. However, photosynthetic organisms are capable of overcoming the toxic effects of light by the rapid and efficient repair of PSII (Aro et al. 1993; Gombos, Wada & Murata 1994; Wada, Gombos & Murata 1994). Therefore, the extent of photoinhibition depends on the balance between photodamage to PSII and the repair of such damage. In the ‘classical’ scheme for the mechanism of photoinhibition, it was assumed that reactive oxygen species (ROS), which are produced by excess light energy, inactivate the photochemical reaction centre of PSII. However, the accumulated evidence now suggests that the main effect of ROS is the inhibition of the repair of photodamaged PSII by, primarily, suppression of the synthesis of proteins de novo (Nishiyama, Allakhverdiev & Murata 2006). Photodamage is probably initiated by the direct effect of light on PSII and, most likely, on the oxygen-evolving complex (Hakala et al. 2005; Ohnishi et al. 2005).

The rate of photodamage to PSII is proportional to the intensity of incident light (or the photon flux density) (Tyystjärvi & Aro 1996; Allakhverdiev & Murata 2004). The rate of repair of photodamaged PSII also depends on the intensity of incident light, but it reaches a maximum under relatively weak light (Allakhverdiev & Murata 2004). The rate of repair is severely depressed by various types of stress, such as oxidative stress (Nishiyama et al. 2001; Allakhverdiev & Murata 2004; Nishiyama et al. 2004), salt stress (Allakhverdiev et al. 2002; Al-Taweel et al. 2007) and low-temperature stress (Allakhverdiev & Murata 2004), with resultant increases in the extent of photoinhibition. Initial studies suggested that oxidative stress might inhibit the elongation of peptides during translation (Nishiyama et al. 2001, 2004). In an analysis of a cyanobacterial translation system in vitro, hydrogen peroxide oxidized elongation factor G (EF-G), causing a conformational change in the factor and, thereby, interrupting translation (Kojima et al. 2007, 2009). Thus, light stress and other types of abiotic stress appear to act synergistically on PSII during photoinhibition (Murata et al. 2007).

The inhibition of the repair of PSII by various types of abiotic stress might be mediated by a mechanism that involves ROS (Takahashi & Murata 2008). Figure 1 shows a hypothetical scheme for the relationships among the transport of electrons, the fixation of CO2 in the Calvin cycle and the inhibition of the synthesis of the D1 protein, which is a key component of PSII. Limitation of the photosynthetic fixation of CO2 decreases the utilization of NADPH, with a resultant decline in the level of NADP+. Since NADP+ is a major acceptor of electrons in photosystem I (PSI), depletion of NADP+ accelerates the transport of electrons to molecular oxygen, with the generation of H2O2 via -O2 (Asada 1999). Consistent with this proposed scheme, interruption of the photosynthetic fixation of CO2 accelerates the production of H2O2, which, in turn, is assumed to inhibit protein synthesis and, thus, the repair of PSII.

image

Figure 1. A hypothetic scheme for the roles of GB in the protection of plants against abiotic stress. When the photosynthetic fixation of CO2 is depressed under abiotic stress, excess electrons from PSI are converted to ROS, which inhibit the repair of photodamaged PSII by inhibiting the synthesis of the pre-D1 protein at the translation step. GB might protect the CO2-fixing enzymes (Rubisco and Rubisco activase) under abiotic stress, thereby sustaining the fixation of CO2, which, in turn, depresses the production of ROS. In addition, GB activates the expression of genes for ROS-scavenging enzymes, which degrade ROS and decrease the levels of ROS in cells, with resultant mitigation of the effects of the abiotic stress on the photosynthetic machinery. GB might also directly protect the translational machinery against abiotic stress and limit the efflux of K+ ions, caused by ROS, either by protecting membrane integrity or by a channel-blocking function.

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A study in Chlamydomonas reinhardtii demonstrated that interruption of the photosynthetic fixation of CO2 by application of exogenous glycolaldehyde, an inhibitor of phosphoribulokinase, or by a missense mutation in the gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (ribulose 1·5-bisphosphate carboxylase/oxygenase; Rubisco) failed to accelerate photodamage to PSII but did inhibit the repair of photodamaged PSII (Takahashi & Murata 2005). In another study with intact chloroplasts from spinach, inhibition of the repair of photodamaged PSII upon interruption of the photosynthetic fixation of CO2 was attributable to inhibition of the synthesis of PSII proteins and, in particular, the D1 protein, at the translational level (Takahashi & Murata 2006).

Heat stress

A recent study with a codA-transgenic cyanobacterium, Synechococcus sp. PCC 7942, which accumulated GB at levels of 60 to 80 mm in the cytosol, demonstrated that the protective effects of GB against the heat-induced inactivation of PSII in darkness are of two types (Allakhverdiev et al. 2007). The codA transgene protected the oxygen-evolving complex, with an upward shift in the temperature for 50% inactivation from 46 to 54 °C, and it protected the photochemical reaction centre, with an upward shift in the temperature for 50% inactivation from 51 to 58 °C. Moreover, an additional protective effect of GB was evident under light-stress conditions. When moderate heat stress, such as incubation of cells at 40 °C, was combined with light stress, PSII was inactivated rapidly, even though each individual stress, when applied separately, inactivated neither the oxygen-evolving complex nor the photochemical reaction centre. Further studies demonstrated that moderate heat stress inhibited the repair of PSII during photoinhibition by interfering with the synthesis of the D1 protein de novo but did not accelerate photodamage directly. The codA transgene and, thus, the accumulation of GB alleviated the inhibitory effect of moderate heat stress on the repair of PSII by accelerating the synthesis of the D1 protein.

Transgenic tobacco plants, which had been transformed with the BADH gene for betaine aldehyde dehydrogenase from spinach, accumulated GB at levels of 0.46 to 4.6 µmol g−1 FW in their chloroplasts (Yang, Liang & Lu 2005). The assimilation of CO2 by the transgenic plants was significantly more tolerant to high temperatures than that of WT plants. Yang et al. (2005) quantified the activity of PSII by measuring chlorophyll fluorescence and the activity of Rubisco by monitoring the incorporation of 14CO2 and they found that the enhanced protection of photosynthesis against high temperatures in transgenic plants was unrelated to the direct activation of PSII, but was caused by enhancement of the activation of Rubisco by Rubisco activase.

At high temperatures, Rubisco activase in stromal fractions became associated with the thylakoid membranes, with a resultant reduction in the extent of activation of Rubisco (Yang et al. 2005). GB appears to support the activation of Rubisco by preventing the sequestration, in thylakoid membranes, of Rubisco activase from soluble stromal fractions and, in this way, it enhances the tolerance of CO2 assimilation to high-temperature stress. These results indicate that the accumulation of GB in vivo leads to the increased thermotolerance of Rubisco activase via prevention of the high temperature-induced association of Rubisco activase with thylakoid membranes (Yang et al. 2005).

Further analysis of these transgenic tobacco plants (Yang et al. 2007) indicated that the accumulation of GB in vivo seemed to reduce the accumulation of ROS during heat stress by maintaining or increasing the activities of ROS-scavenging enzymes (namely, catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase) and, also, by increasing levels of antioxidants, such as ascorbate and reduced glutathione. These results suggest that the accumulation of GB might enhance thermotolerance via suppression of the accumulation of ROS and, also, via the enhanced repair of PSII that has been inactivated by light stress.

Salt stress

Transgenic plants engineered to accumulate GB in vivo exhibit enhanced PSII activity under salt stress. Such plants include transgenic Arabidopsis lines that express, respectively, a bacterial codA gene (Hayashi et al. 1998), the CMO gene from spinach (Hibino et al. 2002) and the ApGSMT and ApSDMT genes from A. halophytica (Waditee et al. 2005); rice plants that express the bacterial codA gene (Sakamoto et al. 1998); and tobacco plants that express the bacterial betA gene from Escherichia coli (Holmstrom et al. 2000) or the BADH gene from spinach (Yang et al. 2008).

Al-Taweel et al. (2007) investigated the effects of salt stress on the repair of PSII and the synthesis of the D1 protein in WT tobacco (N. tabacum cv. Xanthi) and in transformed plants that harboured the katE gene for catalase from E. coli. Salt stress due to NaCl enhanced the photoinhibition of PSII in leaf discs from both WT and katE-transformed plants, but the effects of salt stress were less significant in the transformed plants than in the WT plants. In the presence of lincomycin, an inhibitor of protein synthesis in chloroplasts, the photodamage to PSII occurred at similar rates in leaf discs from both types of plant. These observations suggest that repair of PSII might be protected by catalase. Incorporation of [35S]-Met into the D1 protein during photoinhibition was inhibited by salt stress, and the transformation with the codA gene mitigated this inhibitory effect. Northern blotting revealed that the level of transcripts of the psbA genes, which encode the D1 protein, was not significantly affected by salt stress or by the transformation. These results suggest that salt stress enhanced photoinhibition by inhibiting the repair of PSII and that the katE transgene increased the resistance to salt stress of the translational machinery in chloroplasts.

Yang et al. (2008) examined the effects of salt stress on the growth of seedlings and on photosynthetic activity in WT and transgenic tobacco plants that had been transformed with the BADH gene from spinach. The presence of the BADH transgene resulted in acceleration of the growth of seedlings under salt stress conditions. No significant difference was observed in terms of the accumulation of sodium and chloride ions, leaf water potential and relative water content between WT and transgenic plants. However, salt stress significantly suppressed the assimilation of CO2 and such suppression was less significant in transgenic than in WT plants. Salt stress also decreased the maximal rate of electron transport in PSII and increased the extent of non-photochemical quenching, with less significant changes in transgenic than in WT plants. Salt stress inhibited the activity of Rubisco, fructose 1,6-biphosphatase (FBPase), fructose 1,6-biphosphate aldolase (FBP aldolase) and phosphoribulokinase (PRKase) of chloroplasts, and the extent of inhibition was also less significant in transgenic than in WT plants. Salt stress did not, however, affect the activities of phosphoglycerate kinase, triose phosphate isomerase, ribulose-5-phosphate isomerase, transketolase and sedoheptulose-1,7-biphosphatase in either WT or transgenic plants. These results suggest that the GB-enhanced tolerance of the assimilation of CO2 to salt stress might be one of the physiological bases for the increased tolerance of the growth of transgenic plants to salt stress. Yang et al. (2008) proposed that the accumulation of GB in transgenic plants might stabilize the conformations of Rubisco, FBPase, FBP aldolase, and PRKase and might maintain these enzymes in a functionally active state under salt stress, acting as a molecular chaperone.

Ohnishi & Murata (2006) reported that salt stress due to 0.22 m NaCl repressed the repair of photodamaged PSII by inhibiting the degradation and synthesis of the D1 protein in salt-stressed Synechococcus cells. Deshnium et al. (1995) had previously transformed Synechococcus cells with the codA gene, and they reported that the codA-transformed cells accumulated GB in the cytoplasm at concentrations of 60 to 80 mm. The transformed cells exhibited tolerance to salt stress in terms of growth, the accumulation of chlorophyll and photosynthetic activity, during incubation in 0.4 m NaCl. Salt stress might reduce the integrity and activity of the translational machinery in Synechococcus cells and the synthesis of GB in codA-transformed cells might counteract these effects of salt stress. In addition, salt stress might destabilize and GB might stabilize the proteases, such as FtsH, that are required for the degradation of the D1 protein and other proteins in PSII (Ohnishi & Murata 2006). The above-mentioned results suggest a scenario wherein salt stress might inhibit Rubisco activase or the association of the activase with Rubisco, thereby inhibiting the fixation of CO2. The resultant production of ROS might, in turn, inhibit the repair of PSII. GB might stabilize the association of Rubisco activase with Rubisco and limit the production of ROS under salt-stress conditions (Murata et al. 2007).

Chilling and freezing stress

The exogenous application of GB can protect plants of a variety of species against stress due to freezing or chilling (Chen & Murata 2008). Hayashi et al. (1997) demonstrated that the susceptibility of the oxygen-evolving machinery of PSII to chilling stress was greatly reduced in Arabidopsis plants that had been transformed with a chimeric codA gene for chloroplast-targeted choline oxidase. They found that the extent of photoinhibition after chilling treatment at 5 °C for 4 h under constant light at 250 µmol photons m−2 s−1 was approximately 80% in WT leaves, but was only 45% in leaves from transgenic plants.

Alia et al. (1999) demonstrated that, when leaves from WT Arabidopsis plants that had been grown at 22 °C were incubated at 10 °C in darkness or under low-intensity light (70 µmol photons m−2 s−1) for various periods of time, no obvious inactivation in PSII occurred in leaves from either WT or transgenic plants. When leaves were exposed to high-intensity light (1.2 mmol photons m−2 s−1) at 10 °C and 25 °C, the PSII complex was inactivated in leaves of both types. However, leaves of the transgenic plants were less sensitive than WT leaves to photoinhibition at both temperatures. The photo-induced inactivation of PSII is due, presumably, to damage to the D1 protein, while the repair of the PSII complex includes the degradation of the D1 protein, as well as the synthesis, re-incorporation into the PSII complex, and processing of the precursor of the D1 protein to yield the functional D1 protein.

To investigate whether the protective effects of GB on PSII in vivo might be linked to protein synthesis in chloroplasts at low temperature, Alia et al. (1999) monitored photodamage to PSII in the presence of lincomycin, an inhibitor of protein synthesis in chloroplasts. Under strong light (1.2 mmol photons m−2 s−1) at 10 °C or 25 °C, lincomycin significantly reduced the difference in the extent of photoinhibition between WT and transgenic leaves, whereas lincomycin had no effect on the activity of the PSII complex during incubation of leaves in darkness. Moreover, the effects of temperature on the photodamage to PSII disappeared in both types of leaf. These experiments indicated that the recovery of PSII from photodamage was accelerated by the presence of GB in the chloroplasts of the transgenic plants. When PSII in leaves from WT and transgenic plants was inactivated to various extents by exposure to strong light and was then allowed to recover, PSII in leaves from transgenic plants recovered more rapidly than that in leaves from WT plants. These observations indicate that the step at which GB protects PSII against the combination of light stress and chilling stress is the repair of PSII and, in particular, the repair of the oxygen-evolving complex. It is possible that GB might stabilize Rubisco and reduce the production of ROS at low temperatures (Murata et al. 2007).

Photosystem II of the previously described transgenic Arabidopsis plants also exhibited enhanced tolerance to freezing (Sakamoto et al. 2000). WT and transgenic leaves were frozen to temperatures from 0 to −12 °C, and then warmed to room temperature. The PSII in transgenic leaves remained more active after an episode of freezing to lower temperatures than that in WT leaves.

Osmotic stress

Quan et al. (2004b) transformed maize plants with the betA gene from E. coli and examined the effects of osmotic stress due to 10% (w/v) PEG-6000 on PSII activity at the three-leaf stage. During osmotic-stress treatment at a photon flux density of 1 mmol photons m−2 s−1 for 5 d, the PSII activity in WT plants, as determined by monitoring fluorescence kinetics, was reduced to 75% of the initial activity, while that in transgenic plants was only reduced to 80–85% of the initial activity. The net rate of photosynthesis in WT plants fell to 35% of the initial rate, while that in transgenic plants fell to 40–50% of the initial rate. These results indicate that photosynthetic activity was more stable in the transgenic plants than in the WT plants under osmotic stress.

When osmotic stress is applied to plants, the first target seems to be ATP synthase, and its inactivation leads to a cellular deficit in ATP (Flexas & Medrano 2002; Yokota, Takahara & Akashi 2006). These observations suggest the following possible scenario: osmotic stress inhibits the synthesis of ATP but not the generation of NADPH and, since the fixation of CO2 by Rubisco requires a supply of ATP, the fixation of CO2 ceases and ROS are generated, with resultant inhibition of protein synthesis and of the repair of PSII.

Oxidative stress

Methyl viologen (MV) is an effective generator of ROS. Park et al. (2004, 2007a) demonstrated that GB-accumulating transgenic tomato plants were more tolerant to MV-induced oxidative stress than WT plants. In the presence of MV in moderate light (100 µmol photons m−2 s−1), PSII activity fell significantly in both WT and transgenic plants, although the inactivation of PSII was much slower in transgenic than in WT plants. There was a positive correlation between the level of GB in the chloroplasts and the extent of tolerance to oxidative stress-enhanced photoinhibition, but there was no correlation between the cytosolic level of GB and the extent of tolerance to oxidative stress (Park et al. 2007a).

EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

In the majority of studies of genetically engineered plants that accumulated GB, enzymes for the synthesis of GB were targeted to the chloroplast, whereas, in other studies, the enzymes were targeted to the cytosol (non-targeted), to the mitochondria, or to both the cytosol and the chloroplasts simultaneously (Tables 2 & 3). The effects of the accumulation of GB in chloroplasts have been examined extensively in genetically engineered plants that express a bacterial codA gene whose product is targeted to these organelles (Table 2). Such transgenic plants accumulate GB primarily in their chloroplasts, and they exhibit enhanced tolerance to various abiotic stresses at various developmental stages (Park et al. 2004). Therefore, efforts have been made to determine whether it is the localization of the synthesis of GB in specific subcellular compartments and/or the level of the accumulated GB that determines the extent of such tolerance.

The effects of the subcellular localization of the synthesis and accumulation of GB on stress tolerance were firstly examined in rice plants (Sakamoto et al. 1998). Rice plants exhibit a higher level of tolerance to abiotic stress when they express a chloroplast-targeted choline oxidase and more GB accumulates in the chloroplasts than in the cytosol. The photosynthetic machinery is also better protected against salt stress and cold stress when plants express a chloroplast-targeted codA gene rather than a non-targeted codA gene, whose product is localized mainly in the cytosol, even though the latter plants can accumulate up to five times more GB than the former (Sakamoto et al. 1998).

Park et al. (2004) generated transgenic tomato plants that expressed the bacterial codA gene for choline oxidase that was targeted to chloroplasts. The plants accumulated GB in chloroplasts and exhibited enhanced tolerance to chilling during their entire life cycle, from seed germination to the reproductive stage (Park et al. 2004). Targeting of choline oxidase to the chloroplasts resulted in only very limited accumulation of GB in transgenic leaves (0.09 to 0.30 µmol g−1 FW; Park et al. 2004). The transgenic tomato plants that expressed chloroplast-targeted choline oxidase accumulated up to 86% of the total GB in their chloroplasts, providing the best example of the highest correlation between GB content and the extent of chilling tolerance in terms of the protection of PSII against low temperatures (Park et al. 2004).

Park et al. (2007a) compared transgenic tomato plants in which choline oxidase was targeted to the chloroplasts (Chl-codA lines), to the cytosol (Cyt-codA lines), and to both the chloroplasts and the cytosol simultaneously (ChlCyt-codA lines). The Cyt-codA and ChlCyt-codA lines accumulated up to 5.0- and 6.6-fold more GB, respectively, in their leaves than the Chl-codA lines (0.3 µmol g−1 FW). In an examination of leaves of 5-week-old WT and transgenic seedlings exposed to chilling stress, transgenic plants exhibited stronger tolerance than WT plants. However, Cyt-codA plants with high levels of GB in the cytosol appeared to be less able to protect cells from ion leakage and less able to lower levels of H2O2 than Chl-codA plants. These observations suggest that the accumulation of GB in chloroplasts is more effective in lowering the level of H2O2 and in protecting cells from ion leakage (probably due to cell death) than is the accumulation of GB in the cytosol.

Park et al. (2007a) also compared the responses of the WT and the three types of transgenic tomato plant to chilling and salt stress at the seedling stage and at the germination stage, respectively, as well as the extents of photoinhibition under chilling stress and oxidative stress. For all parameters measured, transgenic plants were generally more tolerant to abiotic stress. The Chl-codA lines exhibited equal (when Fv/Fm and germination rates were compared) or greater (when seedling growth was compared) tolerance to these stresses than the other two types of transgenic plant, even though the level of GB was lowest in the Chl-codA plants. There was a significant correlation between the level of GB in chloroplasts (R2 = 0.50, P < 0.01) and the degree of tolerance to oxidative stress, whereas no correlation was evident between the cytosolic level of GB and stress tolerance, suggesting that even higher levels of GB in chloroplasts might further enhance stress tolerance. Park et al. (2007a) concluded that the accumulation of GB in chloroplasts is the most important parameter in the protection of plants against abiotic stress.

Park, Jeknić & Chen (2006) demonstrated that exogenously applied and incorporated GB was localized mostly in the cytosol, while only a limited portion of the incorporated GB was transported to chloroplasts. Therefore, it is possible that both foliar-applied GB and GB that is synthesized in the cytosol are transported into chloroplasts, but only to a small extent.

In maize plants that had been transformed with a betA gene for cytosol-targeted CDH, the total amount of GB in leaves was as high as 5.7 µmol g−1 FW, which was much higher than that in leaves of WT plants (1.6 µmol g−1 FW) (Quan et al. 2004a,b). However, the concentration of GB in chloroplasts of transgenic and WT maize plants was close to 0.1 µmol g−1 FW, and there was no significant difference between these two types of plant (Quan et al. 2004a). Therefore, Quan et al. (2004a,b) postulated that the enhanced tolerance to chilling and drought stress had resulted from cytosolic GB. The way in which cytosolic GB can protect the photosynthetic activity of chloroplasts remains to be determined.

The accumulation of GB in the mitochondria (Takabe et al. 1998) and peroxisomes (Kishitani et al. 2000) of rice plants also enhances tolerance to abiotic stress. For example, targeting the gene product of a modified betA gene to mitochondria enhanced the tolerance of transgenic rice plants to salt stress (Takabe et al. 1998). Kishitani et al. (2000) reported the introduction of a cDNA of the gene for a peroxisomal BADH from barley into rice. The transgenic rice plants converted high levels of exogenously applied betaine aldehyde to GB and exhibited elevated tolerance to salt, cold and heat stress, maintaining higher than control levels of PSII activity under salt stress. Although the product of the transgene for BADH was targeted to peroxisomes, it was unclear where the synthesis of GB had occurred. If GB is synthesized in peroxisomes, how might it protect PSII in the chloroplasts?

GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

In many crop plants, the reproductive stage is the stage that is most sensitive to abiotic stress, and final yields are seriously diminished if abiotic stress occurs at this stage. Thus, protection against abiotic stress at the reproductive stage is essential for high yields from crop plants in stress-prone areas around the world. Introduction of the biosynthetic pathway of GB into crop plants appears to be an effective strategy for improving tolerance to abiotic stress and increasing yield.

Transgenic Arabidopsis plants engineered to synthesize GB exhibit enhanced tolerance to various kinds of stress during the germination of seeds and the vegetative growth of seedlings (Alia et al. 1998a,b; Hayashi et al. 1998; Alia et al. 1999). Analysis of levels of GB in codA-transgenic Arabidopsis plants (with choline oxidase targeted to chloroplasts) revealed that different organs accumulated different levels of GB, even though the codA gene was driven by a constitutive CaMV 35S promoter (Sulpice et al. 2003). Levels of GB in flowers, siliques and inflorescence apices were approximately five times higher than in leaves. Treatment of WT plants with 100 mm NaCl for 3 d resulted in the abortion of flower buds and decreased numbers of seeds per silique. Microscopic examination of floral structures revealed that salt stress had inhibited the development of anthers, pistils and petals. The production of pollen grains and ovules was also dramatically inhibited. However, in GB-accumulating transgenic plants, these effects of salt stress were significantly reduced. Moreover, transgenic plants produced 21% more siliques and 45% more seeds than WT plants after treatment with 100 mm NaCl for 3 d. Thus, the accumulation of GB in reproductive organs effectively protected the formation of flowers and seeds against salt stress.

Quan et al. (2004a,b) transformed the elite maize inbred line DH4866 with the betA gene for CDH from E. coli. There were no differences in reproductive development between transgenic lines and control (WT) plants in the absence of drought stress. When plants in the field were subjected to drought stress for 3 weeks, the number of pollen grains fell and the formation of ears was delayed in both WT and transgenic plants. However, reproductive development (in terms of numbers of pollen grains and silking time) of transgenic plants was less seriously affected by drought than that of WT plants. After drought treatment for 3 weeks, root biomass, plant height, stem biomass and leaf biomass of transgenic lines were all greater than those of WT plants. The grain weight per plant was 10 to 23% higher in transgenic lines than in WT plants due to larger numbers of grains and larger 1000-grain weights. Thus, the reproductive organs of GB-accumulating transgenic maize plants were less affected by drought stress than those of the WT. It is unknown whether or not the reproductive organs of transgenic maize plants contain elevated levels of GB similar to those found in transgenic Arabidopsis (Sulpice et al. 2003) and tomato (Park et al. 2004, 2007b) plants.

Tomato plants are susceptible to chilling stress, and exposure to temperatures below 10 °C causes various types of damage during plant growth and development. At the flowering stage, chilling reduces pollen viability, and the failure of anthers to release pollen grains results in a substantial reduction in fruit yield. The final yield of tomato plants after an episode of chilling stress depends on the percentage of surviving plants, the number of flowers retained and fruit-set. Park et al. (2004) reported that, when WT and codA-transgenic tomato plants (with choline oxidase targeted to chloroplasts) were chilled at 3 °C for 7 d and then returned to a warm greenhouse, 20% of WT plants died within three or four days whereas almost all the transgenic plants survived. During the first week back in the greenhouse, only 82% of WT flowers were retained, compared to 88 to 100% of codA-transgenic flowers. Moreover, fruit-set was greatly reduced in WT plants as compared to transgenic plants. Overall, GB-accumulating transgenic tomato plants produced an average of 10 to 30% more fruit after chilling. The enhanced tolerance at the reproductive stage might have resulted from the high-level accumulation of GB in reproductive organs. The levels of GB in the petal, pistil, and anther (Park et al. 2004) were 2.8 to 3.8 times higher than those in the leaves of transgenic tomato plants, as observed similarly in codA-transgenic Arabidopsis plants (Sulpice et al. 2003).

GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Many genes related to stress tolerance have been used in attempts to engineer stress tolerance, and these attempts have met with varying success (Park & Chen 2006). However, the resultant transgenic plants often exhibit undesirable phenotypic characteristics, such as growth retardation when plants are grown under non-stress conditions. For example, C-repeat binding factor (CBF)1-transgenic tomato plants exhibit apparent dwarfism, in addition to decreases in fruit-set and in numbers of seeds per fruit (Hsieh et al. 2002a; Lee et al. 2003), and potato plants that overexpress AtCBF genes produce fewer and smaller tubers than WT plants do (Pino et al. 2007). Transformation of Arabidopsis with a dehydration-responsive element-binding (DREB)1a construct and of rice with either a DREB1 construct or the orthologous OsDREB1 construct retarded plant growth in each case (Kasuga et al. 1999; Ito et al. 2006). To minimize such undesirable side effects, it is necessary to use stress-specific promoters, such as the promoter of a gene known as responsive to desiccation 29A (rd29A) and that of the gene for abscisic acid-responsive complex 1 (ABRC1), to ensure the expression of the respective genes under specified environmental conditions (Kasuga et al. 1999; Lee et al. 2003; Pino et al. 2007). Transgenic plants that accumulate high levels of mannitol, sorbitol and trehalose also exhibit improved tolerance to stress but exhibit a variety of undesirable traits under non-stress conditions (Chen & Murata 2002).

To date and to the best of our knowledge, there are no reports of GB-accumulating transgenic plants that exhibit undesirable traits when grown under non-stress conditions. Indeed, some GB-accumulating transgenic plants actually outperform WT plants with respect to yield components. Thus, genetic engineering of the accumulation of GB in transgenic plants not only enhances tolerance to abiotic stress but can also increase yield potential when plants are grown under non-stress conditions, providing unique opportunities for agricultural biotechnology (Chen & Murata 2008).

In early studies, we noted that, under non-stress conditions, the codA transgene, with a chloroplast-targeting sequence, had no negative effects on the germination of seeds (Alia et al. 1998a,b) and the growth of young Arabidopsis plants (Alia et al. 1998a), while it had desirable effects under non-stress conditions, in particular, at reproductive stages. For example, codA-transgenic Arabidopsis plants produced 22% more flowers (and siliques) and 28% more seeds than WT plants when grown under non-stress conditions (Sulpice et al. 2003).

When WT and codA-transgenic tomato plants were grown hydroponically in a greenhouse under normal conditions, there were no visible differences between them in terms of the frequency of germination of seeds and the growth of young plants before the appearance of flower buds (Park et al. 2007b). In contrast to its effects on vegetative organs, the codA transgene clearly increased the sizes of flower buds and flowers of tomato plants under normal conditions (Park et al. 2007b). Increases in size were evident in all the floral organs. In particular, ovaries were unusually large. Individual flowers from transgenic plants weighed approximately twice as much as those from WT plants and the petals were approximately 1.7-fold longer than their WT counterparts. However, the number of petal segments was the same in WT and transgenic plants, and codA-transgenic plants produced approximately 30% fewer flowers per inflorescence than did WT plants. In the absence of hand pollination in the greenhouse, the rate of fruit-set of transgenic plants (42%) was lower than that of WT plants (68%).

The ripe fruits of transgenic tomato plants were considerably larger than those of WT plants, with the ripe fruits from transgenic plants being 54% heavier, on average, than those from WT plants (Park et al. 2007b). Furthermore, ripe transgenic fruits contained six or seven locules, as compared to the two or three locules in fruits of WT plants. However, the number of seeds per transgenic fruit was 62% lower than in WT.

The reproductive organs accumulated the highest levels of GB among the various organs of codA-transgenic Arabidopsis (Sulpice et al. 2003) and tomato (Park et al. 2007b) plants. The flower buds, shoot apices, and locular cavities of ripe transgenic tomato fruits contained GB at levels above 0.5 µmol g−1 FW. Levels of GB in leaves, pericarp and seeds were about half this value, whereas levels of GB in vegetative organs, such as stems, roots and epidermis, were below 0.1 µmol g−1 FW. The enlargement of flowers and fruits might have been related to the high levels of GB in these organs, which were similar to those in Arabidopsis (Sulpice et al. 2003).

GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Under normal growth conditions, plants produce ROS continuously via a variety of metabolic pathways, but they have evolved a variety of enzymatic and non-enzymatic antioxidant systems to prevent ROS from reaching toxic levels (Ashraf 2009; Miller et al. 2010). However, under stress conditions, the amounts of ROS produced in an ‘oxidative burst’ may exceed the capacity of the ROS-scavenging systems, with resultant accumulation of high levels of intracellular ROS. All forms of abiotic stress, including high salt, chilling, freezing and drought, can cause an oxidative burst in plant cells, and the accumulated ROS can cause various types of deterioration and even cell death (Ashraf 2009). Since GB can enhance tolerance to abiotic stress, it seems appropriate to consider the effects of GB on antioxidative defence systems, as they relate to enhanced stress tolerance.

GB stabilizes the oxygen-evolving PSII complex by stimulating its repair when plants are exposed to light and other kinds of stress (see earlier discussion). Moreover, chilling can interfere with the functions of cell membranes, cause the denaturation of proteins, and disturb the electron-transport systems that are embedded in mitochondrial and chloroplast membranes. Then the interruption of electron transport results in the production of ROS. Elevation of intracellular concentrations of ROS can inhibit the synthesis of proteins de novo (Nishiyama et al. 2001; Takahashi & Murata 2008). GB stabilizes membrane integrity against the effects of extreme temperature (Zhao, Aspinal & Paleg 1992; Gorham 1995) and protects complex II in mitochondria (Hamilton & Heckathorn 2001).

Studies in vitro have demonstrated that GB, by itself, does not have antioxidative activity (Smirnoff & Cumbes 1989). Thus, its ROS-scavenging function must be indirect, for example, via the induction of the synthesis or activation of ROS-defence systems. Such a scenario has been demonstrated both after the exogenous application of GB and in transgenic plants that accumulate GB. Hoque et al. (2007) investigated the effects of exogenously applied GB on levels of antioxidants and on the activities of enzymes in the ascorbate-glutathione (ASC-GSH) cycle (Ishikawa & Shigeoka 2008) in tobacco suspension-cultured cells that were exposed to salt stress. They demonstrated that salt stress significantly depressed levels of ASC and GSH, as well as the activities of enzymes in the ASC-GSH cycle, such as ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR). Exogenously applied GB increased the activities of all of these enzymes with the exception of MDHAR. However, GB had no direct effects on the activities of enzymes in the ASC-GSH cycle under normal conditions, only increasing the activities of such enzymes under salt stress. Thus, the GB-enhanced activities of enzymes in the ASC-GSH cycle might protect tobacco cells against salt-induced oxidative stress. Furthermore, salt stress increased the carbonylation of proteins; levels of thiols, disulfides and the reduced (GSH) and oxidized (GSSG) forms of glutathione; and the activities of both glutathione-S-transferase and glyoxylase II. In addition, salt stress altered the redox state of thiol-disulfide and glutathione, and it depressed the activities of glutathione peroxidase and glyoxylase I, which are involved in ROS- and methylglyoxal-detoxification systems (Hoque et al. 2008). Exogenous application of GB resulted in a reduction in protein carbonylation and in increases in the level of the reduced form of glutathione and in the activities of glutathione peroxidase, glutathione-S-transferase and glyoxalase I under salt stress. These results suggest that GB might protect plants against salt-induced dysfunction due to ROS by reducing protein carbonylation and enhancing antioxidant defence and methylglyoxal-detoxification systems (Hoque et al. 2008).

Elevated levels of H2O2 during exposure of plants to chilling or salt stress have often been observed. In Arabidopsis, for example, the level of H2O2 increases two- to three-fold when plants are exposed to chilling or salt stress (Alia et al. 1999). GB-accumulating codA-transgenic Arabidopsis plants are more tolerant to light stress than WT plants, as a consequence of the acceleration of the recovery of PSII from photodamage. It has been proposed that the stress-induced accumulation of H2O2 induces the activation of ROS-scavenging enzymes (Prasad et al. 1994). Alia et al. (1999) analysed the activities of a number of ROS-scavenging enzymes, including catalase (CAT), APX, MDHAR, DHAR, GR, and superoxide dismutase (SOD). Only the activities of CAT and ASP were significantly higher in codA-transgenic Arabidopsis plants than in WT plants. Higher levels of CAT and ASP activities in the transgenic plants might prevent the accumulation of toxic amounts of H2O2.

In GB-accumulating tobacco plants that had been transformed with a gene for BADH from spinach, accumulation of GB increased resistance to heat-enhanced photoinhibition (Yang et al. 2007). Heat stress significantly decreased the activities of CAT, APX, GR, DHAR and MDHAR in WT plants, whereas the activities of these enzymes either decreased considerably, remained unchanged, or even increased in transgenic plants. During heat stress, transgenic plants also contained larger amounts of ascorbate and of the reduced form of glutathione than WT plants. These observations suggest that the accumulation of GB in transgenic plants might depress levels of ROS, which might, in turn, contribute to the increased thermotolerance of transgenic plants.

Treatments that enhance the production of H2O2 also raised levels both of the cat3 transcript and of CAT3 activity, with resultant higher than WT rates of survival and growth upon exposure of maize seedlings to chilling stress (Prasad et al. 1994). Overexpression of the Arabidopsis CBF1 gene (for CRT/DRE-binding factor 1) in tomato plants enhanced tolerance to chilling, oxidative and water stress, and tolerance was associated with elevated levels of catalase activity and of expression of the CATALASE1 (cat1) gene, suggesting that the enhanced tolerance of the transgenic tomato plants might have been due, in large part, to induction of the expression of the cat gene (Hsieh et al. 2002a,b). Exogenous application of GB to WT tomato plants decreased the level of H2O2 and increased the activity of catalase and the level of cat1 transcripts when the plants were exposed to chilling stress (Park et al. 2006). Similar results were also observed when transgenic plants that had been transformed with the codA gene were compared with WT plants (Park et al. 2004). Thus, in tomato plants, it is likely that GB induces an H2O2-mediated antioxidant system, which includes the enhanced expression of CAT genes and the enhanced activity of catalase.

Cuin & Shabala (2007) showed that application of the hydroxyl radical (OH) to Arabidopsis roots resulted in the massive efflux of K+ ions from epidermal cells in the elongation zone. A low concentration (5 mm) of GB significantly reduced the efflux of K+ ions. Since GB has only an indirect effect on the scavenging of free radicals, GB must play some other as-yet-unknown roles in mitigating the negative effects of oxidative stress. Taken together, these observations suggest that: (1) the stress-induced accumulation of ROS is involved in cellular dysfunction; (2) some aspects of GB-enhanced tolerance to stress are due to the role of GB in the activation of ROS-scavenging systems; (3) GB induces the expression of specific genes for ROS-scavenging enzymes (see below); and (4) GB directly protects the plasma membrane, embedded transporters and ion-channel proteins.

GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

As mentioned previously (see also Tables 2 & 3), GB-accumulating transgenic plants with enhanced tolerance to various forms of abiotic stress have been well documented, and various mechanisms have been proposed to explain this enhanced tolerance. However, to date, none of the explanations is entirely satisfactory. One of the major problems is that the level of GB associated with stress tolerance in transgenic plants is quite low. Is it possible that GB, at such low levels, plays a regulatory function in gene expression that leads to increased stress tolerance?

In experiments with GB in vitro, effective concentrations of GB are generally much higher (in the molar range) than in most experiments in vivo. It is possible that, in experiments in vitro, GB might exert its protective effects via a direct effect. By contrast, in experiments in vivo, effective concentrations of GB are in the micromolar range, as are, for example, the effective concentrations of plant hormones (Einset et al. 2007).

Allard et al. (1998) reported that exogenous application of GB increased the freezing tolerance of wheat plants. They examined the accumulation of three low temperature-inducible proteins. One of these proteins, WCOR410, accumulated in the presence of GB, and the final level depended on the concentration of GB. The accumulation of the other two proteins, WCS120 and WCS120, was unaffected by the application of GB. Northern blotting analysis revealed that transcripts of both the WCOR410 and WCOR413 genes accumulated upon exposure of plants to 250 mm GB. Induction of the expression of the low temperature-responsive genes by GB appeared to contribute to the elevated tolerance to freezing. In tomato plants, exogenous application of 1 mm GB increased catalase activity and the level of cat1 transcripts in leaves under non-stress conditions (day 0), with the highest levels recorded on day 1 of chilling stress (Park et al. 2006).

Sakamoto et al. (2000) examined the expression of four cor genes in codA-transgenic Arabidopsis plants to determine whether enhanced tolerance to freezing might be related to the expression of cold-regulated (cor) genes. There were no obvious changes in the expression of the four cor genes (cor15a, cor6.6, cor47 and cor78) in transgenic plants, indicating that, in this case, enhancement of freezing tolerance was not caused by changes in the expression of cor genes. Park et al. (2007b) performed microarray analysis of the expression of genes in flower buds of WT and codA-transgenic tomato and found that the expression of 30 genes was enhanced and that of 29 genes was repressed by the transgene. Exogenous application of GB to tomato leaves also enhanced the expression of a number of genes that are involved in electron transport in chloroplasts and in mitochondria (Park et al. 2006). Chen, Gollop & Heuer (2009) investigated, by a proteomic approach, the effect of exogenously applied GB on the salt stress-induced inhibition of growth in seedlings of tomato ‘Patio’ (a relative salt tolerant cultivar) and ‘F144’ (a salt sensitive cultivar). They found that GB could alleviate the growth inhibition induced by salt stress via changes in levels of six proteins in ‘Patio’ and of two proteins in ‘F144’. The interaction analysis based on computational bioinformatics revealed that major regulatory networks appeared to be PSII, Rubisco, and superoxide dismutase (SOD).

Einset et al. (2007, 2008) demonstrated, in Arabidopsis, that exogenous application of 100 mm GB to both leaves and roots improved tolerance of roots to chilling stress via regulation of the expression of a number of stress-tolerance genes. Such findings suggest new experimental approaches for identifying genes that are involved in the tolerance to chilling stress. Einset et al. (2007) postulated that, at least in part, the GB-enhanced tolerance to abiotic stress might be ascribable to the expression of genes that are important for stress tolerance. Exploiting microarray analysis, they identified 11 ‘GB-up-regulated’ genes in Arabidopsis roots (Einset & Connolly 2009). The GB-up-regulated genes included genes for transcription factors, membrane-trafficking components, ROS-scavenging enzymes and the NADP-dependent ferric reductase of the plasma membrane. Northern blotting analysis revealed that the expression of four genes was significantly enhanced in roots 24 h after the start of treatment with GB (Einset et al. 2007). The products of these genes are membrane-trafficking RabAc4 protein (At5g47960), NADP-dependent ferric reductase FRO2 (At1g01580), mitochondrial catalase 2 (At4g35090) and cell wall peroxidase ATP3a (At 5g64100). Einset et al. (2007) treated WT plants and mutant plants, in which the RabAc4 gene had been disrupted, by applying 100 mm GB to both leaves and roots. Twenty-four hours after the start of this treatment, they subjected the plants to chilling at 4 °C for 48 h and then incubated them under warm conditions for 4 d. WT plants responded strongly to GB in terms of the enhanced growth of shoots and roots, whereas, in mutant plants with the disrupted RabA4C gene, GB did not enhance growth. These authors concluded that RabAc4 is required for the effect of GB on the recovery of plants from chilling-induced damage, and they proposed that the enhanced tolerance to chilling stress was due, at least in part, to gene activation.

In the case of the FRO2 gene for ferric reductase, the FRO-null mutant, frd1-1, failed to respond to GB in chilling assays in terms both of recovery of root growth and of inhibition of the accumulation of ROS (Einset et al. 2008). In WT plants, application of GB had no significant effect on ferric reductase activity. In addition, overexpression of the FRO2 gene did not enhance chilling tolerance. However, the ferric reductase activity in WT and FRO2-overexpressing transgenic lines that had been pre-treated with GB was several-fold higher after chilling than in non-pre-treated controls. These results suggest that (1) FRO2 blocks the accumulation of ROS during chilling and, thus, enhances chilling tolerance; and (2) GB has a major post-transcriptional effect on the activity of FRO2 in the cold. Overall, at least three proteins, namely, RabA4c GTPase (At5g47960), bZIP transcription factor (At3g62420) and FRO2 ferric reductase (At1g01580), are required for GB-induced chilling tolerance (Einset et al. 2007, 2008). Note that, in all three cases, there was a strong association between the accumulation of ROS and chilling stress.

As stated earlier, the production of ROS is closely associated with various types of abiotic stress. The role of ROS-scavenging systems in the GB-mediated tolerance of plant cells to abiotic stress has become more and more important. Indeed, 7 of the 11 genes that were up-regulated in Arabidopsis upon exogenous application of GB are known to encode proteins that are directly involved in scavenging ROS. These proteins include glutathione reductase (At3g24170), cytoplasmic Cu/Zn superoxidase dismutase (At1g08830), glutathione S-transferase (At1g02920), peroxisomal Cu/Zn superoxide dismutase (At5g18100), catalase 2 (At4g35090), monodehydroascorbate reductase (At5g03630), and ascorbate oxidase (At5g21100) (Einset & Connolly 2009). These observations provide strong evidence for the involvement of ROS-scavenging systems in GB-mediated stress tolerance. Einset et al. (2007, 2008) proposed a model to explain how GB-up-regulated genes in roots of Arabidopsis might prevent the accumulation of ROS in cell walls, thereby playing a pivotal role in tolerance to abiotic stress.

Kathuria et al. (2009) produced transgenic indica rice plants that had been transformed with a codA construct for chloroplast-targeted choline oxidase. The transgenic rice plants exhibited increased tolerance to water stress and enhanced detoxification of ROS under water stress. Microarray analysis revealed that at least 50 genes, among the 165 genes whose expression was enhanced more than twofold in transgenic plants, are known to be involved in the responses of plants to abiotic stress. Their study provides strong evidence that the enhanced expression of stress-responsive genes in GB-accumulating transgenic plants might be a plausible explanation for GB-mediated stress tolerance.

Thus, GB, either applied exogenously or synthesized in vivo in transgenic plants, is capable of activating specific genes. The functions of these genes contribute, at least in part, to a reduction in the stress-induced accumulation of ROS. Further studies of the identity of GB-inducible genes and the functions of their products will advance our understanding of the GB-enhanced tolerance of plants to abiotic stress.

CONCLUSIONS AND PERSPECTIVES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES

Various genes have been used to generate transgenic plants that accumulate GB and exhibit enhanced tolerance to abiotic stress. Among such transgenic plants, codA-transgenic plants have been investigated most thoroughly in terms of the physiological and morphological aspects of 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 availability of GB-accumulating transgenic plants has provided tools for investigations of two important aspects of the ability of plants to tolerate abiotic stress. One aspect is the molecular mechanism(s) of the protective effects of GB against various types of abiotic stress. GB protects the photosynthetic machinery and, in particular, PSII against abiotic stress. We still need to identify the most critical steps in the repair of PSII that are affected by individual types of abiotic stress and are protected by GB. The second aspect is the exploration, for agricultural application, of improvements in the productivity of crop plants under stress and non-stress growing conditions. The protective effects of GB on the reproductive organs of plants have been observed in Arabidopsis, maize and tomato. Such protection might involve high levels of GB that accumulate in the reproductive organs as the result of the translocation of GB from other organs, such as leaves.

Based on a recently proposed model for the mechanism of photodamage to PSII (Takahashi & Murata 2008), a hypothetical scheme that explains the protective role of GB against abiotic stress in plant cells is presented in Fig. 1. Possible mechanisms of GB-enhanced tolerance to various types of abiotic stress include: (1) stabilization by GB of the highly ordered structures of certain complex proteins to prevent denaturation when plants or plant cells are exposed to stress conditions; (2) induction by GB of the expression of specific genes that encode ROS-scavenging enzymes and subsequent depression of levels of ROS in plant cells; and (3) prevention by GB of the accumulation of excess ROS, resulting in protection of the photosynthetic machinery from the combined effects of light stress and other kinds of stress, as well as of ion-channel proteins and the integrity of cell membranes.

REFERENCES

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. BIOSYNTHESIS AND ACCUMULATION OF GB
  5. GENETIC ENGINEERING OF THE BIOSYNTHESIS OF GB IN PLANTS
  6. GB PROTECTS THE PHOTOSYNTHETIC MACHINERY AGAINST THE SYNERGISTIC EFFECTS OF LIGHT STRESS AND OTHER TYPES OF ABIOTIC STRESS
  7. EFFECTS OF THE INTRACELLULAR LOCALIZATION OF GB ON TOLERANCE TO ABIOTIC STRESS
  8. GB ENHANCES TOLERANCE OF REPRODUCTIVE ORGANS TO ABIOTIC STRESS
  9. GB-ACCUMULATING TRANSGENIC PLANTS HAVE LARGER REPRODUCTIVE ORGANS AND/OR LARGER NUMBERS OF SEEDS THAN CONTROLS WHEN GROWN UNDER NON-STRESS CONDITIONS
  10. GB ENHANCES ANTIOXIDATIVE DEFENSES IN PLANTS
  11. GB-ENHANCED TOLERANCE TO ABIOTIC STRESS IS ASSOCIATED WITH INDUCTION OF THE EXPRESSION OF SPECIFIC GENES
  12. CONCLUSIONS AND PERSPECTIVES
  13. ACKNOWLEDGMENTS
  14. REFERENCES
  • Ahmad R., Kim M.D., Back K.H., Kim H.S., Lee H.S., Kwon S.Y., Murata N., Chung W.I. & Kwak S.S. (2008) Stress-induced expression of choline oxidase in potato plant chloroplasts confers enhanced tolerance to oxidative, salt, and drought stresses. Plant Cell Reports 27, 687698.
  • Alia, Hayashi H., Chen T.H.H. & Murata N. (1998a) Transformation with a gene for choline oxidase enhances the cold tolerance of Arabidopsis during germination and early growth. Plant, Cell & Environment 21, 232239.
  • Alia, Hayashi H., Sakamoto A. & Murata N. (1998b) Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine. The Plant Journal 16, 155161.
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