Tissue Culture and Related Approaches for Grain Quality Improvement

Gengenbach, B. G.

doi:10.1007/978-94-009-6207-1_8

Cited by 7 publications

(3 citation statements)

References 109 publications

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“…As expected from the fact that lysine and SAM synergistically inhibit AK in many plants (18,26), AEC inhibited AK from wheat suspension cultures even at low concentrations in the presence of SAM (R Kumpaisal et al, unpublished data). Although several AEC-resistant variants are reported in higher plants (10), only a report in Nicotiana sylvestris (16) gives genetic or biochemical evidence to support the reported overproduction of free lysine. The AEC-resistant mutants of N. sylvestris have a feedback insensitive form of DHDPS and unaltered AK (16).…”

Section: Effect Of Inhibitors Various Amino Acids Including Those Ofmentioning

confidence: 98%

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Kumpaisal¹,

Hashimoto²,

Yamada³

1987

Plant Physiol.

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Dihydrodipicolinate synthase, the first enzyme unque to lysine biosynthesis in higher plants, was The biosynthesis of lysine first utilizes the common pathway for the aspartic-family amino acids including lysine, threonine, and methionine, followed by a pathway specific to lysine biosynthesis (5). Since many, though not all, of the enzymes involved in the lysine-specific pathway have been isolated, higher plants are supposed to synthesize lysine via the diaminopimelate pathway (5). In our effort to clarify the regulation of lysine biosynthesis in wheat, we first reported the effects of lysine analogs and aspartate-derived amino acids on growth and AK' (EC. 2.7.2.4) activity in wheat cell suspension cultures (26). The fact that lysine inhibits wheat AK, the first enzyme in the common pathway for aspartate-family amino acids, but not growth of wheat cells, indicates that there should be a more potent site of feedback-inhibition by lysine in the lysine-specific pathway. DHDPS (EC 4.2.1.52), the first enzyme uniquely associated with lysine biosynthesis, catalyzes the condensation of L-ASA and pyruvate. This enzyme activity has been detected in several plants (6,(13)(14)(15)(16)25), and has been found to be localized in the chloroplasts (24). DHDPS from higher plants is feedback-inhibited by low concentrations of lysine (15,16,25) for the enzyme from Escherichia coli (28). For the E. coli enzyme, it is also reported that an enzyme-pyruvate complex exists by Schiff base formation (21) and that the inhibition by lysine is competitive with respect to ASA (20). The kinetic mechanism and the mode of inhibition, however, have not been explored in detail. In view of the potential importance of DHDPS in the regulation of lysine biosynthesis, we studied its properties and kinetics using highly purified enzyme preparations from wheat suspension cultures. MATERILS AND METHODSPlant Material. Suspension cultures of wheat (Triticum aestivum var Chinese Spring), established as described previously (26), were grown on a gyrotory shaker at 100 rpm, 25°C, in the dark. Liquid LS medium (12) supplemented with 10 Mm, 2,4-D, in a total volume of 75 ml, were used in 300-ml flasks. Inoculum weight was 2.0 to 2.5 g fresh weight per flask. Subcultures were carried out at 3-week intervals.Chemicals. Lysine analogs were obtained from the following sources: AEC, DHL, Na-acetyl-L-lysine, Nf-acetyl-L-lysine, and L-lysine hydroxamate were from Sigma Chemical Co.; Ne methyl-L-lysine from Aldrich Chemical Co.; THL from Calbiochem-Behring Corp; and cis and trans isomers of DL-lysyne (A4-lysine) dihydrochloride, DL-lysyne dihydrochloride (26), DL-4-oxalysine dihydrochloride, and DL-homolysine monohydrochloride were synthesized as described previously (26).Butyl-Toyopearl 650 M was obtained from Toyo Soda Manufacturing Co., Tokyo. Sephadex G-25 M, PD-I0 columns, DEAESepharose CL-6B, Phenyl-Superose HR 10/10, Mono Q HR 5/ 5, Superose-12 HR 10/30, and a FPLC system were from Pharmacia, Inc. Protein standards for HPLC were obtained from Oriental...

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Section: Effect Of Inhibitors Various Amino Acids Including Those Ofmentioning

confidence: 98%

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Kumpaisal¹,

Hashimoto²,

Yamada³

1987

Plant Physiol.

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“…Aspartate kinase isoforms in plants are feedback-inhibited by lysine or threonine, and S-adenosyl methionine can increase the inhibition caused by low concentrations of lysine (Bryan 1980;Rognes et al 1980). Growth of cell cultures and seedlings of many plants is inhibited by lysine plus threonine (LT), presumably because of feedback inhibition of AK, and possibly other steps in the pathway causing growth-limiting methionine biosynthesis levels (Dunham and Bryan 1969;Green and Phillips 1974;Gengenbach 1984). Selection for resistance to toxic LT levels, therefore, was proposed as a possible means to obtain mutations of AK that would be less sensitive to feedback regulation (Green and Phillips 1974).…”

Section: Introductionmentioning

confidence: 99%

Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

Dotson

Frisch

Somers

et al. 1990

Planta

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Aspartate kinase (AK; EC 2.7.2.A) catalyzes the first reaction in the biosynthesis pathway for aspartate-derived amino acids in plants. Aspartate kinase was purified from wildtype and two maize (Zea mays L.) genotypes carrying unlinked dominant mutations,Ask LT19 andAsk2 -LT20, that conferred overproduction of threonine, lysine, methionine and isoleucine. The objective of this investigation was to characterize the AKs from mutant and wildtype plants to determine their role in regulating the synthesis of aspartate-derived amino acids in maize. Kernels of the homozygousAsk2 mutant exhibited 174-, 10-, 13- and 2-fold increases in, in this sequence, free threonine, lysine, methionine and isoleucine, compared to wildtype. In wildtype maize, AK was allosterically feedback-inhibited by lysine with 10 μML-lysine required for 50% inhibition. In contrast, AK purified from the isogenic heterozygousAsk and homozygousAsk2 mutants required 25 and 760 μM lysine for 50% inhibition, respectively, indicating thatAsk andAsk2 were separate structural loci for lysine-regulated AK subunits in maize. Further characterization of purified AK from the homozygous mutantAsk2 line indicated altered substrate and lysine inhibition kinetics. The apparent Hill coefficient was 0.7 for the mutantAsk2 AK compared with 1.6 for the wildtype enzyme, indicating that the mutant allele conferred the loss of a lysinebinding site to the mutant AK. Lysine appeared to be a linear noncompetitive inhibitor ofAsk2 AK with respect to MgATP and an uncompetitive inhibitor with respect to aspartate compared to S-parabolic, I parabolic noncompetitive inhibition of wildtype AK. Reduced lysine sensitivity of theAsk2 gene product appeared to reduce the lysine inhibition of all of the AK activity detected in homozygousAsk2 plants, indicating that maize AK is a heteromeric enzyme consisting of the two lysine-sensitive polypeptides derived from theAsk andAsk2 structural genes.

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“…Resistant cell lines to AEC have been isolated in maize and potato, but without modification of the lysine content (13,20). AEC-tolerant Pennisetum accumulated free lysine 5 to 7 times that of the control in vegetative tissues, but no genetic or biochemical evidence was available (3).…”

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confidence: 99%

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

Frankard

Ghislain²,

Jacobs³

1992

Plant Physiol.

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Lysine and threonine overproducer mutants in Nicotiana sylvestris, characterized by an altered regulation of, respectively, dihydrodipicolinate synthase and aspartate kinase activities, were crossed to assess the effects of the simultaneous presence of these genes on the biosynthesis of aspartate-derived amino acids. The monogenic dominant behavior of both resistance traits was confirmed, and their loci were found to be unlinked.

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Tissue Culture and Related Approaches for Grain Quality Improvement

Cited by 7 publications

References 109 publications

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Purification and Characterization of Dihydrodipicolinate Synthase from Wheat Suspension Cultures

Lysine-insensitive aspartate kinase in two threonine-overproducing mutants of maize

Two Feedback-Insensitive Enzymes of the Aspartate Pathway in Nicotiana sylvestris

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