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First published online August 23, 2002; 10.1105/tpc.001701 American Society of Plant Biologists Silencing of Phosphoethanolamine N-Methyltransferase Results in Temperature-Sensitive Male Sterility and Salt Hypersensitivity in ArabidopsisInstitute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China 3 To whom correspondence should be addressed. E-mail jyli{at}genetics.ac.cn; fax 86-10-64873428
S-Adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT; EC 2.1.1.103) catalyzes the key step in choline (Cho) biosynthesis, the N-methylation of phosphoethanolamine. Cho is a vital precursor of the membrane phospholipid phosphatidylcholine, which accounts for 40 to 60% of lipids in nonplastid plant membranes. Certain plants use Cho to produce the osmoprotectant glycine betaine, which confers resistance to salinity, drought, and other stresses. An Arabidopsis mutant, t365, in which the PEAMT gene is silenced, was identified using a new sense/antisense RNA expression system. t365 mutant plants displayed multiple morphological phenotypes, including pale-green leaves, early senescence, and temperature-sensitive male sterility. Moreover, t365 mutant plants produced much less Cho and were hypersensitive to salinity. These results demonstrate that Cho biosynthesis not only plays an important role in plant growth and development but also contributes to tolerance to environmental stresses. The temperature-sensitive male sterility caused by PEAMT silencing may have a potential application in agriculture for engineering temperature-sensitive male sterility in important crop plants.
Choline (Cho) is a key metabolite in plants because it is needed to synthesize the major membrane lipid phosphatidylcholine (PC), which accounts for 40 to 60% of lipids in nonplastid plant membranes (Moore, 1990  ; Bolognese and McGraw, 2000). Studies have shown that the synthesis of PC is affected by the plant growth regulator indole-3-acetic acid (Price-Jones and Harwood, 1983), suggesting that PC has a fundamental function in plant growth and development. Evidence also has indicated that freezing tolerance is correlated with changes in the amount and degree of polyunsaturation of PC (Sikorska and Kacperska-Palacz, 1980; Kinney et al., 1987). In addition, it has been shown that salinity stress induces an increase in the turnover of PC in Arabidopsis suspension-cultured cells (Pical et al., 1999), suggesting that PC also may play an important role in plant responses to salt and other environmental stresses.
In certain plants, such as spinach, Cho is oxidized to glycine betaine (GlyBet) by chloroplast enzymes (Rhodes and Hanson, 1993
Although the engineered plants have shown increased stress tolerance, the improvements in stress tolerance are relatively small because the levels of GlyBet obtained by this engineering strategy are low (Nuccio et al., 1999
Cho biosynthesis has been investigated in diverse plants. Evidence from these investigations indicated that Cho can be produced via three parallel, interconnected pathways involving sequential methylations of an ethanolamine moiety at the free base, phospho-base (P-base), and phosphatidyl-base (Ptd-base) levels (Rhodes and Hanson, 1993
The three methylation steps at the P-base level from phosphoethanolamine to phosphocholine all are catalyzed by the cytosolic enzyme S-adenosyl-L-methionine:phosphoethanolamine N-methyltransferase (PEAMT; EC 2.1.1.103), and the first methylation step exerts major control over flux through the entire pathway (Datko and Mudd, 1988a Despite the importance of PEAMT as the regulatory enzyme in the biosynthesis of Cho moieties, it remains unclear how PEAMT affects plant growth and development and the plant response to environmental stresses. Considering the fundamental functions of Cho biosynthesis, a null mutation is likely lethal. No such mutants have been isolated to date. In this study, we report the characterization of an Arabidopsis transgenic mutant, t365, which was identified using a new sense/antisense RNA expression (SARE) system developed to determine gene functions on a genome scale, and cloning of the gene responsible for the t365 mutant phenotype. The T365 gene encodes a PEAMT, which catalyzes all three methylation steps required to convert phosphoethanolamine to phosphocholine. Expression of the T365 transgene causes silencing of endogenous PEAMT and results in temperature-sensitive male sterility, decrease in Cho production, and salt hypersensitivity in Arabidopsis plants.
A SARE System To introduce sense or antisense RNA into Arabidopsis plants, we first designed a plant expression vector,  455, by replacing the central stuffer of GEM-12 with the plasmid pJL453 (Figure 1). 455 contains the colE1 replicon, a conditional mini-RK2 replicon, and the gene encoding  -lactamase, which allow replication and selection in Escherichia coli and Agrobacterium tumefaciens. The vector also carries the T-DNA border sequences for the transfer and integration of cDNA into a plant genome, the double 35S promoter of Cauliflower mosaic virus (2x35S) and the nopaline synthase (NOS) terminator for in planta expression of the cDNA, and the neomycin phosphotransferase gene for the selection of transgenic plants. The plasmid portion of 455 is flanked by two lox direct repeats, which are used for site-specific recombination under catalysis of the cre protein (Sternberg et al., 1986; Elledge et al., 1991).
After infecting E. coli strain trpC9830( KC) (Li et al., 1995) cells that produce cre protein, a precise excision occurred in 455 between the lox sites, resulting in a binary plasmid. Through this recombination system, a cDNA library constructed in 455 could be converted into a library of binary plasmids, which then could be transformed into plants by Agrobacterium. After transformation, the cDNA inserts introduced into transgenic plants were isolated by PCR using primers designed to anneal to the sequences of the 35S promoter and the NOS terminator. The genes that were disrupted by the T-DNA insertion could be identified through plasmid rescue or inverse PCR.
Transgenic Plants Generated by the SARE System
For this experiment, Among these 600 lines, 19 were found to exhibit apparent morphological abnormalities, including decreased plant size, altered leaf shape, bushy inflorescence, and reduced fertility (Figures 2A to 2J; Table 1, t189, t104, t77, t101, t113, t30, t307, t44, and t365). Mutants with early flowering time, short flower pedicels, and propendent siliques also were identified (Figures 2K and 2L; Table 1, t59 and t414). Genetic analyses of the T3 seeds from the 19 mutant lines showed that they were all capable of transmitting the mutant phenotypes to their progeny (data not shown), indicating that the phenotypes are heritable.
Morphology of t365 Mutant Plants To validate the SARE system in identifying gene functions, one of the transgenic mutant lines, t365, was chosen for in-depth analysis. When grown under continuous light at 22°C, t365 rosette leaves were pale green in juvenile stages (Figures 3A and 3B) and senesced early in the late reproductive stage (Figures 3C and 3D). Compared with the wild type (Figure 3E), the t356 mutant plants had shorter siliques and produced fewer seeds (Figure 3F). Microscopic examination of wild-type and t365 flowers revealed that t365 mutant plants produced less pollen than the wild type (Figures 3G and 3H), suggesting that the reduced fertility may be caused by abnormal male fertility in t365 mutant plants.
To test this possibility, we conducted cross-pollination experiments. Cross-pollination of t365 mutant plants with wild-type pollen resulted in normal seed set. In reciprocal crosses, using pollen from t365 mutant plants to pollinate wild-type stigmas, fewer seeds were produced (data not shown). Thus, the reduced seed set observed in t365 mutant plants was caused by a decrease in male fertility.
Genetic Analysis of the t365 Mutant
To determine which plants harbored the T-DNA insert, PCR was used to amplify the DNA samples from the individual T2 plants. A PCR product was detected in all of the 70 t356-looking plants and in 18 of the 47 wild-type-looking plants. This result was confirmed by DNA gel blot analysis using the 35S promoter DNA as a probe (data not shown). The segregation ratio of 88 to 29 fits the expectation for a single-locus insertion of the transgene (
Cloning of the T365 Gene
T365 Encodes a PEAMT in Arabidopsis
The t365 Mutant Phenotype Results from Silencing of the Endogenous PEAMT
To determine further if there were PEAMT transcripts in t365 mutant plants, we used reverse transcriptase–PCR to detect the transcripts. Although the amount was significantly lower than that in wild-type plants (Figure 4B, lane 1) and t365 wild-type-looking plants (Figure 4B, lane 2), the transcripts did exist in t365 mutant plants (Figure 4B, lane 3). Therefore, it is likely that the amount of the PEAMT transcripts in the wild-type-looking plants was sufficient to support normal plant growth and development, whereas the residual level of PEAMT transcripts in t365 mutant plants only supported plant survival. To confirm that the t365 mutant phenotype was caused by suppression of the endogenous PEAMT by the T365 transgene, the 745-bp cDNA fragment isolated from t365 transgenic plants was cloned into vector pJL453-2 (Figure 1) in both the sense and antisense orientations and retransformed into Arabidopsis. Of 13 and 11 independent sense and antisense transgenic lines, 6 and 4 lines showed morphology similar to that of t365, as shown in Figure 5A. The same effect on the endogenous PEAMT transcripts was observed in these transgenic lines as in t365 (Figure 5B).
Besides the original t365 mutant, independent antisense and sense-suppressed lines were used for further temperature-sensitive male sterility and saline sensitivity studies (see below). All of the phenotypes of the original t365 mutant also were observed in the independent antisense and sense-suppressed lines. These results demonstrate that the t365 mutant phenotypes are caused by silencing of the endogenous PEAMT rather than by disruption of a gene function caused by the T-DNA insertion.
Silencing of PEAMT Leads to a Decrease in Cho Content in t365 Mutant Plants
t365 Mutant Plants Show Temperature-Sensitive Male Sterility In plants, PEAMT catalyzes the committing methylation step, the methylation of phosphoethanolamine to phosphomonomethylethanolamine, and the subsequent two methylation steps at the P-base level for de novo PC biosynthesis (Nuccio et al., 2000 ; McNeil et al., 2001). Deficiency in the two terminal phospholipid N-methyltransferase activities required for de novo biosynthesis of PC in Saccharomyces cerevisiae opi3 mutant strains leads to a temperature-sensitive growth phenotype (McGraw and Henry, 1989). To investigate the influence of temperature on the phenotype of t365 mutant and independent sense/antisense transgenic plants, we grew them under continuous light at 20, 23, and 26°C, respectively. Although temperature had no effect on the pale-green color of t365 leaves, it had a dramatic effect on the fertility of t365 mutant plants. When grown at 20°C, t365 mutant plants produced a lot of seeds (Figure 7B), although fewer than wild-type plants (Figure 7A). By contrast, t365 mutant plants grown at both 23 and 26°C showed markedly decreased fertility. No seeds were produced in the abnormal siliques (Figures 7C and 7D). Compared with wild-type plants (Figure 7E), t365 mutant plants grown at temperatures of 23°C or greater had diminished pollen production (Figures 7G and 7H), which confirmed that the decreased fertility under these growth conditions was caused by male sterility. The similar temperature-sensitive male sterility phenotypes of sense/antisense transgenic plants also were observed, as shown in Figure 8.
t365 Mutant Plants Are Hypersensitive to Salt Stress After conversion from phosphoethanolamine through the three methylation steps, phosphocholine is either incorporated into PC or metabolized to Cho (Summers and Weretilnyk, 1993 ; McNeil et al., 2000). Some plants such as spinach have chloroplast enzymes that catalyze the two-step oxidation of Cho to GlyBet, which has strong osmoprotectant properties and confers tolerance to salinity and other stresses (Rhodes and Hanson, 1993; Gorham, 1995; Sakamoto and Murata, 2000). Arabidopsis lacks the genes for Cho oxidation (McNeil et al., 2000, 2001). However, evidence has shown that changes in the amount of PC are correlated with salt stress (Kinney et al., 1987; Pical et al., 1999), suggesting that PC may be involved in the response to high salinity in plant cells.
To determine if the silencing of PEAMT in t365 mutant plants has an effect on their response to salinity, we performed a series of salt stress experiments with different concentrations of NaCl. Figure 9A shows the phenotype of soil-grown t365 mutant plants after treatment with 200 mM NaCl. Compared with wild-type plants (Figure 9A, left), t365 mutant plants showed a hypersensitivity phenotype (Figure 9A, right). The sensitivity also was tested on plates containing 100 mM NaCl, as described previously (Liu and Zhu, 1998
To confirm that the salt hypersensitivity was caused by the decrease in Cho content as a result of the silencing of PEAMT in t365 mutant plants, Cho was supplied to the medium containing 100 mM NaCl. The salt inhibition to t365 mutant plants was ameliorated significantly (Figure 9D, right). These results demonstrate that the silencing of PEAMT in t365 mutant plants results in hypersensitivity to high salinity.
Here, we have described a system based on SARE in transgenic plants for the identification of gene functions on a genome scale. Using this system, we identified 19 transgenic mutants with apparent abnormal morphological phenotypes from the first 600 transgenic lines. We systematically characterized one of these mutants, t365, and cloned the gene responsible for the t365 mutant phenotypes. The T365 gene encodes a PEAMT that catalyzes the three methylation steps required to convert phosphoethanolamine to phosphocholine in de novo Cho or PC biosynthesis. Expression of a T365 transgene in t365 mutant plants causes silencing of the endogenous PEAMT, which leads to abnormal morphological phenotypes, temperature-sensitive male sterility, reduction in Cho biosynthesis, and salt hypersensitivity.
In plants, PC is a dominant constituent of membrane phospholipids and is necessary for a wide array of structural and biochemical functions (Moore, 1990
Under certain growth conditions, opi3 mutants produce membranes virtually devoid of PC (McGraw and Henry, 1989 Temperature mainly affects the fertility of t365 mutant plants. When grown at low temperatures, such as 20°C, t365 mutant plants are fertile and can produce some seeds. However, at temperatures greater than 23°C, t365 mutant plants showed dramatically decreased fertility, and no seeds were produced in the abnormal siliques. Cross-pollination experiments indicated that the sterility observed in t365 mutant plants is attributable to failure to produce functional pollen. The ability to induce male sterility in t365 mutant plants by growth at high temperatures suggests that silencing of PEAMT may provide an efficient biotechnological approach to engineer temperature-sensitive male sterility in agriculturally important plants.
Evidence has shown that salinity and hyperosmotic stress induce dramatic increases in the levels of phosphatidylinositol 4,5-bisphosphate and diacylglycerol pyrophosphate and also affect the turnover of PC (Sikorska and Kacperska-Palacz, 1980
In plants, it has been shown that PEAMT is the key enzyme of Cho biosynthesis. Overexpression of PEAMT in transgenic tobacco increases the levels of phosphocholine by 5-fold and free Cho by 50-fold without affecting PC content or plant growth (McNeil et al., 2001 Plants have evolved various protective mechanisms to acclimate themselves to unfavorable environments for continued survival and growth. Cho biosynthesis is one such mechanism. However, to date, the precise function of the Cho biosynthesis pathway in stress tolerance has not been well studied as a result of the lack of mutants of the Cho biosynthesis enzymes. In t365 mutant plants, expression of one of the Cho biosynthesis enzymes, PEAMT, is suppressed, allowing us to determine the roles of the Cho biosynthesis pathway in plant stress tolerance.
Successful isolation of the t365 mutant and cloning of the T365 gene demonstrated the applicability of the SARE system. This system has several advantages. First, the cre-lox site-specific recombination system used to convert the
The SARE system is based on the expression of sense or antisense RNA in transgenic plants, which has been demonstrated to suppress the expression of an endogenous gene or genes with sequence homology (van der Krol et al., 1988
Plant Growth Arabidopsis thaliana plants were grown on vermiculite saturated with 0.3 x B5 medium under continuous illumination (80 to 120 µE·m-2·s-1) at 23°C as described previously (Mou et al., 2000 ) or under the conditions specified. For temperature treatment, plants were germinated and grown in three Versatile Environmental Test Chambers (Sanyo, Tokyo, Japan) under continuous illumination (100 µE·m-2·s-1) at 20, 23, and 26°C, respectively. For salt stress treatment, 2-week-old soil-grown plants were watered with 50, 100, 200, and 400 mM NaCl. The phenotype was examined 10 days after NaCl treatment. To determine salt sensitivity on plates, seedlings were grown on Murashige and Skoog (1962) nutrient (GIBCO) agar plates for 3 days and then transferred to vertical Murashige and Skoog (1962) agar plates supplemented with 100 mM NaCl. The phenotype was examined 10 days after transfer to 100 mM NaCl.
Construction of
Construction of an Arabidopsis cDNA Library in
Automatic Conversion of the Phage cDNA Library into a Binary Plasmid Library and Generation of Transgenic Plants
The plasmid DNA was extracted and purified with polyethylene glycol as described (Sambrook et al., 1989
Arabidopsis Col-0 plants were transformed with these agrobacteria via vacuum infiltration (Bechtold et al., 1993
Mutant Screening and Genetic Analysis
Isolation of the T365 Gene The 5' sequence of the T365 cDNA was obtained by rapid amplification of cDNA ends–PCR (RACE-PCR) with a 5'-RACE Kit (Boehringer Mannheim). The three specific primers used in the RACE reactions were SP1 (5'-GTTTGTGAGCACTGGTGGAC-3'), SP2 (5'-TGGCCAAAGACACGCTCATAG-3'), and SP3 (5'-GACATAAGC-TCCAATGCACTTG-3'). The PCR products were cloned into pBluescript II SK+/- and sequenced as described above.
Confirmation That the T365 Transgene Leads to the t365 Mutant Phenotype
RNA Gel Blot and Reverse Transcriptase–PCR Analyses
For reverse transcriptase–PCR analysis, 2 µg of total RNAs was used in a 20-µL reverse transcription reaction as described (Li et al., 1998
Determination of Choline Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
We thank Xinnian Dong (Duke University) and Wendy E. Durrant (Duke University) for comments on the manuscript. The gift of Agrobacterium strain GV3101(pMP90RK) from Csaba Koncz and Jeff Schell (Max-Plank-Institut für Zuchtungsforschung, Koln, Germany) is much appreciated. This research was supported by grants from the State Transgenic Plant Program and the State Hi-Tech Program (863), by the National Natural Science Foundation of China Grant 39889003, and by a National Distinguished Young Scholar Award to J.L.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.001701.
1 These two authors contributed equally to this work.
2 Current address: Developmental, Cell, and Molecular Biology Group, Department of Biology, Box 91000, Duke University, Durham, NC 27708-1000. Received January 24, 2002; accepted May 21, 2002.
Alia, Hayashi, H., Sakamoto, A., and Murata, N. (1998). Enhancement of the tolerance of Arabidopsis to high temperatures by genetic engineering of the synthesis of glycinebetaine. Plant J. 16, 155–161.[CrossRef][ISI][Medline] Alia, Kondo, Y., Sakamoto, A., Nonaka, H., Hayashi, H., Saradhi, P.P., Chen, T.H., and Murata, N. (1999). Enhanced tolerance to light stress of transgenic Arabidopsis plants that express the codA gene for a bacterial choline oxidase. Plant Mol. Biol. 40, 279–288.[CrossRef][ISI][Medline]
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402. Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated gene transfer by infiltration of Arabidopsis thaliana plants. C. R. Acad. Sci. Paris 316, 1194–1199. Becker, I. (1990). Construction of Chimeric Phosphoenolpyruvate Carboxylase Gene and Its Expression in Potato. PhD dissertation (Aachen, Germany: Rheinisch Westfälische Technische Hochschule).
Bolognese, C.P., and McGraw, P. (2000). The isolation and characterization in yeast of a gene for Arabidopsis S-adenosylmethionine:phospho-ethanolamine N-methyltransferase. Plant Physiol. 124, 1800–1813.
Datko, A.H., and Mudd, S.H. (1988a). Enzymes of phosphatidyl-choline synthesis in Lemna, soybean and carrot. Plant Physiol. 88, 1338–1348.
Datko, A.H., and Mudd, S.H. (1988b). Phosphatidylcholine synthesis. Plant Physiol. 88, 854–861.
Elledge, S.J., Mulligan, J.T., Ramer, S.W., Spottswood, M., and Davis, R.W. (1991). Lambda YES: A multifunctional cDNA expression vector for the isolation of genes by complementation of yeast and Escherichia coli mutations. Proc. Natl. Acad. Sci. USA 88, 1731–1735.
Elmayan, T., Balzergue, S., Beon, F., Bourdon, V., Daubremet, J., Guenet, Y., Mourrain, P., Palauqui, J.C., Vernhettes, S., Vialle, T., Wostrikoff, K., and Vaucheret, H. (1998). Arabidopsis mutants impaired in cosuppression. Plant Cell 10, 1747–1758.
Furner, I.J., Sheikh, M.A., and Collett, C.E. (1998). Gene silencing and homology-dependent gene silencing in Arabidopsis: Genetic modifiers and DNA methylation. Genetics 149, 651–662. Gorham, J. (1995). Betaines in higher plants: Biosynthesis and role in stress metabolism. In Amino Acids and Their Derivatives in Higher Plants, R.M. Wallsgrove, ed (Cambridge, UK: Cambridge University Press), pp. 171–203. Hayashi, H., Alia, Mustardy, L., Deshnium, P., Ida, M., and Murata, N. (1997). Transformation of Arabidopsis thaliana with the codA gene for choline oxidase: Accumulation of glycinebetaine and enhanced tolerance to salt and cold stress. Plant J. 12, 133–142.[CrossRef][ISI][Medline]
Hofgen, R., Axelsen, K.B., Kannangara, C.G., Schuttke, I., Pohlenz, H.D., Willmitzer, L., Grimm, B., and von Wettstein, D. (1994). A visible marker for antisense mRNA expression in plants: Inhibition of chlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase antisense gene. Proc. Natl. Acad. Sci. USA 91, 1726–1730.
Holmstrom, K.O., Somersalo, S., Mandal, A., Palva, T.E., and Welin, B. (2000). Improved tolerance to salinity and low temperature in transgenic tobacco producing glycine betaine. J. Exp. Bot. 51, 177–185. Hu, Y., Bao, F., and Li, J. (2000). The promotive effect of brassinolide on cell division involves a distinct cycD3-induction pathway in Arabidopsis. Plant J. 24, 693–710.[CrossRef][ISI][Medline]
Huang, J., Hirji, R., Adam, L., Rozwadowski, K.L., Hammerlindl, J.K., Keller, W.A., and Selvaraj, G. (2000). Genetic engineering of glycinebetaine production toward enhancing stress tolerance in plants: Metabolic limitations. Plant Physiol. 122, 747–756. Jorgensen, R. (1990). Altered gene expression in plants due to trans interactions between homologous genes. Trends Biotechnol. 8, 340–344.[CrossRef][ISI][Medline] Kinney, A.J., Clarkson, D.T., and Loughman, B.C. (1987). The regulation of phosphatidylcholine biosynthesis in rye (Secale cereale) roots: Stimulation of the nucleotide pathway by low temperature. Biochem. J. 242, 755–759.[ISI][Medline] Koncz, C., and Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383–396.[CrossRef][ISI]
Li, H., Wu, G., Ware, D., Davis, K.R., and Yang, Z. (1998). Arabidopsis Rho-related GTPases: Differential gene expression in pollen and polar localization in fission yeast. Plant Physiol. 118, 407–417. Li, J., Zhao, J., Rose, A.B., Schmidt, R., and Last, R.L. (1995). Arabidopsis phosphoribosylanthranilate isomerase: Molecular genetic analysis of triplicate tryptophan pathway genes. Plant Cell 7, 447–461.[Abstract]
Liu, J., and Zhu, J.K. (1998). A calcium sensor homolog required for plant salt tolerance. Science 280, 1943–1945.
Martienssen, R.A. (1998). Functional genomics: Probing plant gene function and expression with transposons. Proc. Natl. Acad. Sci. USA 95, 2021–2026.
McGraw, P., and Henry, S.A. (1989). Mutations in the Saccharomyces cerevisiae opi3 gene: Effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis. Genetics 122, 317–330.
McNeil, S.D., Nuccio, M.L., Rhodes, D., Shachar-Hill, Y., and Hanson, A.D. (2000). Radiotracer and computer modeling evidence that phospho-base methylation is the main route of choline synthesis in tobacco. Plant Physiol. 123, 371–380.
McNeil, S.D., Nuccio, M.L., Ziemak, M.J., and Hanson, A.D. (2001). Enhanced synthesis of choline and glycine betaine in transgenic tobacco plants that overexpress phosphoethanolamine N-methyltransferase. Proc. Natl. Acad. Sci. USA 98, 10001–10005. Moore, T.S. (1990). Biosynthesis of phosphatidylinositol. Inositol Metab. Plant 9, 107–112.
Mou, Z., He, Y., Dai, Y., Liu, X., and Li, J. (2000). Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology. Plant Cell 12, 405–417.
Mudd, S.H., and Datko, A.H. (1989a). Synthesis of EA and its regulation in Lemna paucicostata. Plant Physiol. 91, 587–597.
Mudd, S.H., and Datko, A.H. (1989b). Synthesis of methylated EA moieties: Regulation by Cho in Lemna. Plant Physiol. 90, 296–305.
Mudd, S.H., and Datko, A.H. (1989c). Synthesis of methylated EA moieties: Regulation by Cho in soybean and carrot. Plant Physiol. 90, 306–310. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant. 15, 473–497.[CrossRef] Nuccio, M.L., Rhodes, D., McNeil, S.D., and Hanson, A.D. (1999). Metabolic engineering of plants for osmotic stress resistance. Curr. Opin. Plant Biol. 2, 128–134.[CrossRef][ISI][Medline] Nuccio, M.L., Russell, B.L., Nolte, K.D., Rathinasabapathi, B., Gage, D.A., and Hanson, A.D. (1998). The endogenous choline supply limits glycine betaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J. 16, 487–496.[CrossRef][ISI][Medline]
Nuccio, M.L., Ziemak, M.J., Henry, S.A., Weretilnyk, E.A., and Hanson, A.D. (2000). cDNA cloning of phosphoethanolamine N-methyltransferase from spinach by complementation in Schizosaccharomyces pombe and characterization of the recombinant enzyme. J. Biol. Chem. 275, 14095–14101.
Pical, C., Westergren, T., Dove, S.K., Larsson, C., and Sommarin, M. (1999). Salinity and hyperosmotic stress induce rapid increases in phosphatidylinositol 4,5-bisphosphate, diacylglycerol pyrophosphate, and phosphatidylcholine in Arabidopsis thaliana cells. J. Biol. Chem. 274, 38232–38240. Price-Jones, M.J., and Harwood, J.L. (1983). Hormonal regulation of phosphatidylcholine synthesis in plants: The inhibition of cytidylyltransferase activity by indol-3-ylacetic acid. Biochem. J. 216, 627–631.[Medline]
Prud'homme, M.-P., and Moore, T.S. (1992a). Phosphatidylcholine synthesis in castor bean endosperm: Free bases as intermediates. Plant Physiol. 100, 1527–1535.
Prud'homme, M.-P., and Moore, T.S. (1992b). Phosphatidylcholine synthesis in castor bean endosperm: The occurrence of an S-adenosyl-L-methionine:ethanolamine N-methyltransferase. Plant Physiol. 100, 1536–1540. Rhodes, D., and Hanson, A.D. (1993). Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 357–384.[CrossRef][ISI]
Sakamoto, A., and Murata, N. (2000). Genetic engineering of glycinebetaine synthesis in plants: Current status and implications for enhancement of stress tolerance. J. Exp. Bot. 51, 81–88.
Sakamoto, A., and Murata, N. (2001). The use of bacterial choline oxidase, a glycinebetaine-synthesizing enzyme, to create stress-resistant transgenic plants. Plant Physiol. 125, 180–188. Sakamoto, A., Valverde, R., Alia, Chen, T.H., and Murata, N. (2000). Transformation of Arabidopsis with the codA gene for choline oxidase enhances freezing tolerance of plants. Plant J. 22, 449–453.[CrossRef][ISI][Medline] Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Sikorska, E., and Kacperska-Palacz, A. (1980). Frost-induced phospholipid changes in cold-acclimated and nonacclimated rape leaves. Physiol. Plant. 48, 201–206. Sternberg, N., Sauer, B., Hoess, R., and Abremski, K. (1986). Bacteriophage P1 cre gene and its regulatory region: Evidence for multiple promoters and for regulation by DNA methylation. J. Mol. Biol. 187, 197–212.[CrossRef][ISI][Medline] Summers, P., and Weretilnyk, E.A. (1993). Choline synthesis in spinach in relation to salt stress. Plant Physiol. 103, 1269–1276.[Abstract] van Blokland, R., van der Geest, N., Mol, J.N.M., and Kooter, J.M. |
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Abstract
INTRODUCTION
2 x 105 independent transformants were obtained. Transformants were allowed to grow on plates for 3 days and then were pooled and cultured for 2 h at 28°C. Arabidopsis Col-0 wild-type plants were transformed with these agrobacteria via vacuum infiltration (Bechtold et al., 1993
2 = 0.036, P > 0.80). However, the segregation of 70 t356-looking to 47 wild-type-looking plants did not fit any simple Mendelian ratio. 
