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Andrew D. Hanson - One of the best experts on this subject based on the ideXlab platform.

  • Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco.
    Plant physiology, 2000
    Co-Authors: Scott D. Mcneil, David Rhodes, Brenda L. Russell, Michael L. Nuccio, Yair Shachar-hill, Andrew D. Hanson
    Abstract:

    Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach Choline Monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with Choline (Cho). We therefore used metabolic modeling in conjunction with [14C]Cho labeling experiments and in vivo 31P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [14C]Cho doses used were large enough to markedly perturb Cho and phosphoCholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast transport limitation corrected, additional engineering interventions would still be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design.

  • Choline import into chloroplasts limits glycine betaine synthesis in tobacco: analysis of plants engineered with a chloroplastic or a cytosolic pathway.
    Metabolic engineering, 2000
    Co-Authors: Michael L. Nuccio, Andrew D. Hanson, Scott D. Mcneil, Michael J. Ziemak, Ravinder K. Jain, Gopalan Selvaraj
    Abstract:

    Abstract The biosynthesis of the osmoprotectant glycine betaine (GlyBet) is a target for metabolic engineering to enhance stress resistance in crops. Certain plants synthesize GlyBet in chloroplasts via a two-step oxidation of Choline (Cho). In previous work, a chloroplastic GlyBet synthesis pathway was inserted into tobacco (which lacks GlyBet) by expressing spinach Choline Monooxygenase (CMO). The transformants had low CMO enzyme activity, and produced little GlyBet (⩽70 nmol g −1 fresh wt). In this study, transfor- mants with up to 100-fold higher CMO activity showed no further increase in GlyBet. In contrast, tobacco expressing a cytosolic GlyBet synthesis pathway accumulated significantly more GlyBet (430 nmol g −1 fresh wt), suggesting that subcellular localization influences pathway flux. Modeling of the labeling kinetics of Cho metabolites observed when [ 14 C]Cho was supplied to engineered plants demonstrated that Cho import into chloroplasts indeed limits the flux to GlyBet in the chloroplastic pathway. A high-activity Cho transporter in the chloroplast envelope may therefore be an integral part of the GlyBet synthesis pathway in species that accumulate GlyBet naturally, and hence a target for future engineering.

  • Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth
    Plant physiology, 1998
    Co-Authors: Brenda L. Russell, Bala Rathinasabapathi, Andrew D. Hanson
    Abstract:

    Choline Monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach ( Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet ( Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl 2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

  • The endogenous Choline supply limits glycine betaine synthesis in transgenic tobacco expressing Choline Monooxygenase
    The Plant journal : for cell and molecular biology, 1998
    Co-Authors: Michael L. Nuccio, Brenda L. Russell, Bala Rathinasabapathi, Kurt D. Nolte, Douglas A. Gage, Andrew D. Hanson
    Abstract:

    Summary Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of Choline. Introducing GlyBet accumulation into plants that lack it is a well-established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by Choline Monooxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous Choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02– 0.05 μmol g–1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM Choline or phosphoCholine, their Choline and GlyBet levels increased by at least 30-fold. The Choline precursors mono- and dimethylethanolamine also enhanced Choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to Choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with Choline-oxidizing genes, the size of the free Choline pool and the metabolic flux to Choline need to be increased to attain GlyBet levels as high as those in natural accumulators.

  • Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach.
    Plant physiology, 1995
    Co-Authors: Michael Burnet, P.j. Lafontaine, Andrew D. Hanson
    Abstract:

    The osmoprotectant glycine betaine is synthesized via the path-way Choline -> betaine aldehyde -> glycine betaine. In spinach (Spinacia oleracea), the first step is catalyzed by Choline Monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO. [14C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [14C]betaine aldehyde to [14C]glycine betaine, which is isolated by ion exchange. The assay was used in the purification of CMO from leaves of salinized spinach. The 10-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on Choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about 600-fold and recovery of activity was 0.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a polypeptide with a molecular mass of 45 kD. Taken with the value of 98 kD estimated for native CMO (R. Brouquisse, P. Weigel, D. Rhodes, C.F. Yocum, A.D. Hanson [1989] Plant Physiol 90: 322-329), this indicates that CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein.

Teruhiro Takabe - One of the best experts on this subject based on the ideXlab platform.

  • Proline, Glycinebetaine, and Trehalose Uptake and Inter-Organ Transport in Plants Under Stress
    Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants, 2019
    Co-Authors: Suriyan Cha-um, Vandna Rai, Teruhiro Takabe
    Abstract:

    Proline, glycinebetaine, and trehalose function as compatible solutes and are upregulated in plants under abiotic stress. The uptake and inter-organ transport in plants are largely unknown. We review the current information on the transport of these osmoprotectants under abiotic stress. Proline metabolism involves several subcellular compartments. Proline concentrations are regulated by the interplay of biosynthesis, degradation, and transport processes. Among the proline transporter proteins, both general amino acid permeases and selective compatible solute transporters were identified. The review summarized our current knowledge on proline transport under abiotic stress conditions. Trehalose is a nonreducing disaccharide formed by two glucose molecules. Sugar transporters have essential roles in the appropriate distribution of carbohydrates throughout the plants. Trehalose transporter has been poorly characterized because it is difficult to predict the characteristics of sugar transporters based solely on the amino acid sequences. Transport properties of exogenous applied trehalose for abiotic stress tolerance have been discussed. Glycinebetaine is synthesized by two-step oxidations of Choline with enzymes Choline Monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Different biosynthetic pathways among monocot and dicot plants were discussed. Expression and substrate specificity of betaine/proline transporters from various plants were compared. Exogenous application of glycinebetaine to plants under stress conditions improved abiotic stress tolerance and gained some attentions. Further application using the important plants both in laboratory and field will contribute to increase the crop production under stress environments.

  • Suppressed expression of Choline Monooxygenase in sugar beet on the accumulation of glycine betaine
    Plant physiology and biochemistry : PPB, 2015
    Co-Authors: Nana Yamada, Yoshito Tanaka, Hiroyuki Takahashi, Kunihide Kitou, Kosuke Sahashi, Hideto Tamagake, Teruhiro Takabe
    Abstract:

    Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of Choline. Choline Monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of Choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from Choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of Choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation.

  • Differential accumulation of glycinebetaine and Choline Monooxygenase in bladder hairs and lamina leaves of Atriplex gmelini under high salinity
    Journal of plant physiology, 2014
    Co-Authors: Koichi Tsutsumi, Yoshito Tanaka, Nana Yamada, Suriyan Cha-um, Teruhiro Takabe
    Abstract:

    Atriplex gmelini is a halophyte and possesses bladder hairs on the leaf surface. It is also known to accumulate the osmoprotectant glycinebetaine (GB). However, it remains unclear whether GB and its biosynthetic enzyme Choline Monooxygenase (CMO) accumulate in the bladder hairs. Microscopic observation of young leaves showed many bladder hairs on their surfaces, but their total number decreased along with leaf maturity. Sodium Green fluorescent approach revealed Na(+) accumulation in bladder cells of young leaves when A. gmelini was grown at high salinity (250 mM NaCl). Due to fewer bladder hairs in mature leaves, Na(+) accumulation was mostly found in mesophyll cells of mature leaves under high salinity. GB accumulation was found at significant level in both bladder- and laminae-cells without any addition of NaCl and its content increased at high salinity. CMO was not found in bladder hairs or young leaf laminae. Instead, the CMO protein expression was observed in mature leaves and that showed increased accumulation with increasing concentration of NaCl. Furthermore, in situ hybridization experiments revealed the expression of a transporter gene for GB, AgBetT, in the bladder hairs. Based on these results, the synthesis and translocation of GB in A. gmelini were discussed.

  • Preferential accumulation of betaine uncoupled to Choline Monooxygenase in young leaves of sugar beet--importance of long-distance translocation of betaine under normal and salt-stressed conditions.
    Journal of plant physiology, 2009
    Co-Authors: Nana Yamada, Takashi Hibino, Yoshito Tanaka, Hideto Tamagake, Worrawrat Promden, Koji Yamane, Teruhiro Takabe
    Abstract:

    It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of Choline in which Choline Monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied Choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.

  • Regulation of betaine synthesis by precursor supply and Choline Monooxygenase expression in Amaranthus tricolor
    Journal of experimental botany, 2007
    Co-Authors: Nazmul H. Bhuiyan, Takashi Hibino, Nana Yamada, Akira Hamada, Vandna Rai, Teruhiro Takabe
    Abstract:

    In plants, betaine is synthesized upon abiotic stress via Choline oxidation, in which Choline Monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect abiotic stress, it is shown here that an exogenous supply of precursors such as Choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt stress, but not under normal conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of A. tricolor CMO (AmCMO) and betaine was much slower in roots than in leaves, and a transient accumulation of proline was observed in the roots. Antisense expression of AmCMO mRNA suppressed the saltinduced accumulation of AmCMO and betaine, but increased the level of Choline ;2– 3-fold. This indicates that betaine synthesis is highly regulated by AmCMO expression. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated. Deletion analysis of the AmCMO promoter region revealed that the 410 bp fragment upstream of the translation start codon contains the sequence responsive to salt stress. These data reveal that the promoter sequence of CMO, in addition to precursor supply, is important for the accumulation of betaine in the betaine-accumulating plant A. tricolor.

Susumu Takio - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt
    Plant Cell Reports, 2015
    Co-Authors: Elsayed Mohamed, Ryuya Matsuda, Ahmed A. El-khatib, Katsuaki Takechi, Hiroyoshi Takano, Susumu Takio
    Abstract:

    Key message Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. Abstract The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S . maritima is related to European and India S . maritima , while Japanese S . maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S . maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S . maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding Choline Monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S . maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

  • Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt.
    Plant cell reports, 2015
    Co-Authors: Elsayed Mohamed, Ryuya Matsuda, Ahmed A. El-khatib, Katsuaki Takechi, Hiroyoshi Takano, Susumu Takio
    Abstract:

    Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding Choline Monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

Elsayed Mohamed - One of the best experts on this subject based on the ideXlab platform.

  • Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt
    Plant Cell Reports, 2015
    Co-Authors: Elsayed Mohamed, Ryuya Matsuda, Ahmed A. El-khatib, Katsuaki Takechi, Hiroyoshi Takano, Susumu Takio
    Abstract:

    Key message Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. Abstract The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S . maritima is related to European and India S . maritima , while Japanese S . maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S . maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S . maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding Choline Monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S . maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

  • Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt.
    Plant cell reports, 2015
    Co-Authors: Elsayed Mohamed, Ryuya Matsuda, Ahmed A. El-khatib, Katsuaki Takechi, Hiroyoshi Takano, Susumu Takio
    Abstract:

    Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding Choline Monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

Brenda L. Russell - One of the best experts on this subject based on the ideXlab platform.

  • Metabolic modeling identifies key constraints on an engineered glycine betaine synthesis pathway in tobacco.
    Plant physiology, 2000
    Co-Authors: Scott D. Mcneil, David Rhodes, Brenda L. Russell, Michael L. Nuccio, Yair Shachar-hill, Andrew D. Hanson
    Abstract:

    Previous work has shown that tobacco (Nicotiana tabacum) plants engineered to express spinach Choline Monooxygenase in the chloroplast accumulate very little glycine betaine (GlyBet) unless supplied with Choline (Cho). We therefore used metabolic modeling in conjunction with [14C]Cho labeling experiments and in vivo 31P NMR analyses to define the constraints on GlyBet synthesis, and hence the processes likely to require further engineering. The [14C]Cho doses used were large enough to markedly perturb Cho and phosphoCholine pool sizes, which enabled development and testing of models with rates dynamically responsive to pool sizes, permitting estimation of the kinetic properties of Cho metabolism enzymes and transport systems in vivo. This revealed that import of Cho into the chloroplast is a major constraint on GlyBet synthesis, the import rate being approximately 100-fold lower than the rates of Cho phosphorylation and transport into the vacuole, with which import competes. Simulation studies suggested that, were the chloroplast transport limitation corrected, additional engineering interventions would still be needed to achieve levels of GlyBet as high as those in plants that accumulate GlyBet naturally. This study reveals the rigidity of the Cho metabolism network and illustrates how computer modeling can help guide rational metabolic engineering design.

  • Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth
    Plant physiology, 1998
    Co-Authors: Brenda L. Russell, Bala Rathinasabapathi, Andrew D. Hanson
    Abstract:

    Choline Monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach ( Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet ( Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl 2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

  • The endogenous Choline supply limits glycine betaine synthesis in transgenic tobacco expressing Choline Monooxygenase
    The Plant journal : for cell and molecular biology, 1998
    Co-Authors: Michael L. Nuccio, Brenda L. Russell, Bala Rathinasabapathi, Kurt D. Nolte, Douglas A. Gage, Andrew D. Hanson
    Abstract:

    Summary Certain plants produce glycine betaine (GlyBet) in the chloroplast by a two-step oxidation of Choline. Introducing GlyBet accumulation into plants that lack it is a well-established target for metabolic engineering because GlyBet can lessen damage from osmotic stress. The first step in GlyBet synthesis is catalyzed by Choline Monooxygenase (CMO), a stromal enzyme with a Rieske-type [2Fe-2S] center. The absence of CMO is the primary constraint on GlyBet production in GlyBet-deficient plants such as tobacco, but the endogenous Choline supply is also potentially problematic. To investigate this, we constructed transgenic tobacco plants that constitutively express a spinach CMO cDNA. The CMO protein was correctly compartmented in chloroplasts and was enzymatically active, showing that its [2Fe-2S] cluster had been inserted. Salinization increased CMO protein levels, apparently via a post-transcriptional mechanism, to as high as 10% of that in salinized spinach. However, the GlyBet contents of CMO+ plants were very low (0.02– 0.05 μmol g–1 fresh weight) in both unstressed and salinized conditions. Experiments with [14C]GlyBet demonstrated that this was not due to GlyBet catabolism. When CMO+ plants were supplied in culture with 5 mM Choline or phosphoCholine, their Choline and GlyBet levels increased by at least 30-fold. The Choline precursors mono- and dimethylethanolamine also enhanced Choline and GlyBet levels but ethanolamine did not, pointing to a major constraint on flux to Choline at the first methylation step in its synthesis. The extractable activity of the enzyme mediating this step in tobacco was only 3% that of spinach. We conclude that in GlyBet-deficient plants engineered with Choline-oxidizing genes, the size of the free Choline pool and the metabolic flux to Choline need to be increased to attain GlyBet levels as high as those in natural accumulators.

  • Choline Monooxygenase an unusual iron sulfur enzyme catalyzing the first step of glycine betaine synthesis in plants prosthetic group characterization and cdna cloning
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Bala Rathinasabapathi, Michael Burnet, Brenda L. Russell, Douglas A. Gage, Paochi Liao, Gordon J Nye, Paul Scott, John H Golbeck
    Abstract:

    Plants synthesize the osmoprotectant glycine betaine via the route Choline → betaine aldehyde → glycine betaine. In spinach, the first step is catalyzed by Choline Monooxygenase (CMO), a ferredoxin-dependent stromal enzyme that has been hypothesized to be an oligomer of identical subunits and to be an Fe-S protein. Analysis by HPLC and matrix-assisted laser desorption ionization MS confirmed that native CMO contains only one type of subunit (Mr 42,864). Determination of acid-labile sulfur and nonheme iron demonstrated that there is one [2Fe-2S] cluster per subunit, and EPR spectral data indicated that this cluster is of the Rieske type—i.e., coordinated by two Cys and two His ligands. A full-length CMO cDNA (1,622 bp) was cloned from spinach using a probe generated by PCR amplification for which the primers were based on internal peptide sequences. The ORF encoded a 440-amino acid polypeptide that included a 60-residue transit peptide. The deduced amino acid sequence included two Cys-His pairs spaced 16 residues apart, a motif characteristic of Rieske-type Fe-S proteins. Larger regions that included this motif also showed some sequence similarity (≈40%) to Rieske-type proteins, particularly bacterial oxygenases. Otherwise there was very little similarity between CMO and proteins from plants or other organisms. RNA and immunoblot analyses showed that the expression of CMO in leaves increased several-fold during salinization. We conclude that CMO is a stress-inducible representative of a new class of plant oxygenases.