Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Brears, Timothy; Liu, Christopher; Knight, Thomas J.; Coruzzi, Gloria M.

doi:10.1104/pp.103.4.1285

Cited by 78 publications

(54 citation statements)

References 17 publications

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“…In a strategy to determine whether NH4+-dependent AS activity could be enhanced in plants, Brears et al (1993) created a site-specific mutation in the pea AS1 cDNA that specifically deleted the three amino acids required for Gln binding. When this construct driven by the cauliflower mosaic virus 35s promoter was introduced into the heterologous host tobacco, two independent lines of transgenic tobacco showing overexpression of the mutated Gln-dependent AS continued to show high levels of Asn.…”

Section: Discussionmentioning

confidence: 99%

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

et al. 1997

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Asparagine, the primary assimilation product from N2 fixation in temperate legumes and the predominant nitrogen transport product in many plant species, is synthesized via asparagine synthetase (AS; EC 6.3.5.4). Here, we report the isolation and characterization of a cDNA and a gene encoding the nodule-enhanced form of AS from alfalfa. The AS gene is comprised of 13 exons separated by 12 introns. The 5' flanking region of the AS gene confers nodule-enhanced reporter gene activity in transformed alfalfa. This region also confers enhanced reporter gene activity in dark-treated leaves. These results indicate that the 5' upstream region of the AS gene contains elements that affect expression in root nodules and leaves. Both AS mRNA and enzyme activity increased -10-to 20-fold during the development of effective nodules. lneffective nodules have strikingly reduced amounts of AS transcript. Alfalfa leaves have quite low levels of AS mRNA and protein; however, exposure to darkness resulted in a considerable increase in both. In situ hybridization with effective nodules and P-glucuronidase staining of nodules from transgenic plants showed that AS is expressed in both infected and uninfected cells of the nodule symbiotic zone and in the nodule parenchyma. RNA gel blot analysis and in situ hybridization results are consistent with the hypothesis that initial AS expression in nodules is independent of nitrogenase activity.

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Section: Discussionmentioning

confidence: 99%

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

et al. 1997

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“…The ASN1 cDNA previously isolated from Arabidopsis (Lam et al, 1994) was subcloned into a plant expression vector (pTEV; Brears et al, 1993) and was placed immediately downstream from the cauliflower mosaic virus (CaMV) 35S promoter. After transformation into Agrobacterium tumefaciens GV3101/pMP90 (Koncz and Schell, 1986), the target ASN1 gene was subsequently integrated into the genome of Arabidopsis ecotype Columbia-0 (Col-0) using vacuum infiltration techniques (Bechtold and Pelletier, 1993).…”

Section: Construction Of Transgenic Arabidopsis Overexpressing Asn1mentioning

confidence: 99%

“…The ASN1 cDNA (Lam et al, 1994) was subcloned into a pTEV vector (Brears et al, 1993) and expressed under the control of the CaMV 35S promoter. Binary vector constructions were transferred into the disarmed Agrobacterium tumefaciens strain GV3101/pMP90 (Koncz and Schell, 1986) by electroporation.…”

Section: Generation Of 35s-asn1 Linesmentioning

confidence: 99%

“…Previous studies from our laboratory examined the effects of altering the expression of the pea AS1 gene in transgenic tobacco (Nicotiana tabacum). Although expressing the pea AS1 gene ectopically in tobacco led to an increase in levels of free Asn in vegetative tissues, the effects on nitrogen status in sink tissues were not thoroughly analyzed (Brears et al, 1993).…”

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Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

et al. 2003

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In wild-type Arabidopsis, levels of ASN1 mRNA and asparagine (Asn) are tightly regulated by environmental factors and metabolites. Because Asn serves as an important nitrogen storage and transport compound used to allocate nitrogen resources between source and sink organs, we tested whether overexpression of the major expressed gene for Asn synthetase, ASN1, would lead to changes in nitrogen status in the ultimate storage organ for metabolites-seeds. Transgenic Arabidopsis constitutively overexpressing ASN1 under the cauliflower mosaic virus 35S promoter were constructed (35S-ASN1). In seeds of the 35S-ASN1 lines, three observations support the notion that the nitrogen status was enhanced: (a) elevations of soluble seed protein contents, (b) elevations of total protein contents from acid-hydrolyzed seeds, and (c) higher tolerance of young seedlings when grown on nitrogen-limiting media. Besides quantitative differences, changes in the relative composition of the seed amino acid were also observed. The change in seed nitrogen status was accompanied by an increase of total free amino acids (mainly Asn) allocated to flowers and developing siliques. In 35S-ASN1 lines, sink tissues such as flowers and developing siliques exhibit a higher level of free Asn than source tissues such as leaves and stems, despite significantly higher levels of ASN1 mRNA observed in the source tissues. This was at least partially due to an enhanced transport of Asn from source to sink via the phloem, as demonstrated by the increased levels of Asn in phloem exudates of the 35S-ASN1 plants.In higher plants, nitrogen is acquired from the environment via nitrate reduction (Crawford and Arst, 1993), ammonia uptake, or nitrogen fixation (Burris and Roberts, 1993). All inorganic nitrogen must first be reduced to ammonia by the action of nitrate and nitrite reductase before being assimilated into amino acids Miflin and Lea, 1980;Lea et al., 1990). In plants, the majority of ammonia is assimilated into organic form via the combined action of Gln synthetase and Gln 2-oxoglutarate aminotransferase. The products of Gln synthetase-Gln 2-oxoglutarate aminotransferase cycle, Gln, and Glu are highly reactive and are used in various anabolic pathways. Through the action of Asn synthetase (AS; EC 6.3.5.4), Gln will react with Asp to form Asn and Glu. Because all the substrates and products of the AS-catalyzed reaction are major nitrogen carriers in plant metabolism for transporting nitrogen in the phloem, the AS enzyme is believed to play an important role in regulating the flow of nitrogen into the organic nitrogen pool (Lam et al., 1994). Asn is an especially important nitrogen transport amino acid because it has a high nitrogen to carbon ratio (relative to the other amide amino acid Gln) and is relatively inert compared with the other nitrogen-transporting amino acids (Glu, Asp, and Gln). Therefore, Asn can be used for long-range nitrogen transport and storage, which is vital to physiological processes such as germination and nitrogen assimilation Siecie...

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“…Preparation of Transgenic A. thaliana-To constitutively express the wild type and mutant OsGAP1 proteins in A. thaliana, the recombination constructs were subcloned into a binary vector (V7) (21) and expressed under the control of the cauliflower mosaic virus 35S promoter before being transformed into the wild type Arabidopsis (Col-0) via a vacuum infiltration method (22) using the Agrobacterium tumefaciens strain GV3101 (pMP90) (23). Screening of the T1 seeds was performed on MS agar plates supplemented with 50 mg/liter kanamycin.…”

Section: Expression and Purification Of Mbp Fusion Proteins Of The Wimentioning

confidence: 99%

Site-directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 for the Physiological Functions of the Rice GTPase-activating Protein 1 (OsGAP1)

Yung

Cheung

Miao

et al. 2015

Journal of Biological Chemistry

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Background: OsGAP1, a plant-specific C2-domain protein, binds to phospholipids and an unconventional G-protein (OsYchF1). Results: We solved the OsGAP1 structure and performed site-directed mutagenesis to identify two functional surfaces for binding to phospholipids versus OsYchF1. Conclusion:Interactions with phospholipids and OsYchF1 play important roles in the functions of OsGAP1. Significance: Our data advance the understanding of the structure-function relationship of C2-domain proteins.

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Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco

Cited by 78 publications

References 17 publications

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Nitrogen assimilation in alfalfa: isolation and characterization of an asparagine synthetase gene showing enhanced expression in root nodules and dark-adapted leaves.

Overexpression of the ASN1 Gene Enhances Nitrogen Status in Seeds of Arabidopsis

Site-directed Mutagenesis Shows the Significance of Interactions with Phospholipids and the G-protein OsYchF1 for the Physiological Functions of the Rice GTPase-activating Protein 1 (OsGAP1)

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