The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.

/pdf/tryptophan-mutants-in-arabidopsis-the-consequences-of-13tyqogs58.pdf

Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes.

INTRODUCTION.88 DESCRIPTION OF THE PATHWAY.88 Reactions Regulation of Metabolite Flow 9 Feedback inhibition.8 Repression of enzyme synthesis.8 Early Evolutionary Speculations 90 ENTEROBACTERIACEAE. Operon Structure. go Genetic studies 9 Messenger RNA studies.91 Regulatory mutations Polarity and antipolarity 2 Deviations from Coordinacy.93 Subunit Structure of Enzymes and Amino Acid Sequences ....... ............. 93 Organisms Unable to Synthesize Tryptophan.95 OTHER GRAM-NEGATIVE BACTIERIA.9 Pseudomonadaceae.95 Multiple operon trp gene arrangement.95 Subunit structure of enzymes and amino acid sequences.96 Regulation of enzyme synthesis. 7 Acinetobacter.9 Multiple operon arrangement of genes.97 Subunit structure of enzymes.98 Regulation of enzyme synthesis GRAM-POSITIVE BACTERIA.............................. 99 Bacillus and Clostridium 99 Chromosomal arrangement Subunit structure of enzymes and amino acid sequences .100 Regulation of enzyme synthesis.10 Staphylococcus and Micrococcus 1 Chromosomal arrangement.10 Subunit structure and regulation of enzymes.101 Streptomyces.101 Chromosomal arrangement.101 BACTERIA STUDIED NON-GENETICALLY.102 Chromobacterium, Lactobacillus, and Blue-Green Bacteria.102 Subunit structure and regulation of enzymes.10 EUKARYOTIC ORGANISMS.102 Fungi.10 Chromosomal arrangement.102 Subunit structure of enzymes 3 Regulation of enzyme synthesis.104 Algae, Euglenoids, and Plants 5 Subunit structure ofenzymes.105 Regulation of enzyme synthesis.105 GENERAL CONCLUSIONS ABOUT EVOLUTION OF THEPATHWAY ........ 105 Conservation of Reaction Mechansims.105 Variation in Chromosomal Disposition of Genes.106 Translocation and gene fusion.10 Regulatory mechanisms.1 6 Enzymes Shared with Other Pathways.107 NATURAL RELATIONSHIPS REVEALED BY COMPARATIVE STUDIES ...... 107 Amino Acid Sequences .....I.. .. . . ......... . . ....I..................107 Nucleotide Sequences and Nucleic Acid Hybridization .. . 108

Gene rearrangements in the evolution of the tryptophan synthetic pathway.

Experimentally determined lethal temperatures and temperatures limiting growth or reproduction in the life histories of 15 benthic algal species were used to infer possible phytogeographic boundaries in the North Atlantic Ocean. These appeared to correspond closely with phytogeographic boundaries based on distribution data. Many boundaries appeared to be of a composite nature. For instance, the southern boundary ofNemalion helminthoides is interpreted as a “southern reproduction boundary” on the N. Atlantic E. shore and a “southern lethal boundary” on the N. Atlantic W. shore. The northern boundary on both sides of the ocean is a “northern reproduction boundary”.N. helminthoides is a typical representative of the “amphiatlantic temperate distribution group”, to which seven other of the fifteen investigated species belong (Chondrus crispus, Desmarestia aculeata, D. viridis, Monostroma grevillei, Acrosiphonia “arcta” with a comparable composite southern boundary;Rhodochorton purpureum with a “southern lethal boundary”).Polysiphonia ferulacea andDictyota dichotoma are treated as representatives of the “amphiatlantic tropical-to-warm-temperate distribution group”, andP. denudata as representative of the “amphiatlantic tropical-to-temperate group”.P. harveyi belongs to the N.E. American temperate group and is bounded by a “northern reproduction boundary” and a “southern reproduction boundary”. This is one of the very few species endemic to N.E. America. This poor endemism is ascribed to the vast adverse sediment shores and their additional acting as barriers to glacial northsouth displacements of the flora; it is not related to the wide annual temperature fluctuations (>20 °C) typical for N.E. America. The temperate algal flora of Japan, however, which is extremely rich in endemic species is subject to equally wide annual temperature fluctuations.Bonnemaisonia hamifera is such a Japanese endemic, which has been accidentally introduced into the North Atlantic Ocean where its life history seems to be disrupted: it is maintained mainly by vegetative propagation of the heteromorphic tetrasporophyte. The species of the “warm temperate Mediterranean-Atlantic group” are probably too stenothermous for life on N.E. American shores; they need annual temperature fluctuations<20 °C.Acrosymphyton purpuriferum seems to belong to this group, but arguments are presented to unite this species withA. caribaeum and to range it under the “amphiatlantic tropical-to-warm-temperate group”.Clathromorphum circumscriptum belongs to the “Arctic distribution group” and has a “southern reproduction boundary” across the ocean along the 3 °C February isotherm. This species is able to survive temperatures of about 20 °C. Five amphiequatorial temperate species discussed in this paper and four in another related paper have similar maximum winter temperatures of 14–17 °C (mean monthly values) allowing reproduction. Their amphiequatorial distribution can be explained by assuming similar low temperatures in the euphotic zone along E. Pacific and E. Atlantic equatorial coasts i.e. in narrow inshore belts of intensified upwelling during the presumably intensified glacial circulation of the ocean gyres.

/pdf/phytogeographic-distribution-groups-of-benthic-marine-algae-4ig2fyuv3z.pdf

Phytogeographic distribution groups of benthic marine algae in the North Atlantic Ocean. A review of experimental evidence from life history studies

The field of amino acid biosynthesis from inorganic nitrogen has been one of the most active in plant biochemistry in the last five years and a recent book has been devoted entirely to it (Miflin 1980 a). To provide a reasonable coverage of the field in a short chapter is difficult; here we present an outline of the biochemical pathways involved in the flow of nitrogen from ammonia into (and in some cases out of) amino acids, and give some indication of how the pathways may be regulated and how the reactions are integrated into the plant’s metabolism, particularly in relation to subcellular localization. We have attempted to support this outline with a reasonable number of citations from which the interested reader can derive much further information; nevertheless the citations are by no means exhaustive and to the authors of relevant papers that have been omitted we offer our regrets and ask for their understanding.

Ammonia Assimilation and Amino Acid Metabolism

Publisher Summary This chapter discusses aromatic amino acid biosynthesis and its regulation. The multibranched shikimic acid pathway provides the precursors for synthesis of the three common protein amino acids—phenylalanine, tyrosine, and tryptophan—in both microorganisms and higher plants. Secondary metabolites such as alkaloids, coumarins, flavonoids, lignin precursors, indole derivatives, and other phenolic compounds arise from this pathway. The pathway of aromatic amino acid biosynthesis is common to all organisms capable of de novo synthesis. Shikimate kinase enzyme catalyzes the phosphorylation of shikimate to yield shikimate 3-phosphate. Tryptophan biosynthesis diverges from the common precursors of phenylalanine and tyrosine with the conversion of chorismate to anthranilate. The formation of anthranilate proceeds with an elimination of the enolpyruvyl side chain of chorismate accompanied by a glutamine donated amidotransfer. Regulation of biosynthesis in the multibranched shikimic acid pathway relies heavily on feedback mechanisms. The shikimate pathway enzymes exist in the chloroplast and also in the cytoplasm.

Aromatic Amino Acid Biosynthesis and Its Regulation

The occurrence of tryptophan synthetase in excised tomato root cultures (Lycopersicon esculentum Mill. var. Red River) and pea seedlings (Pisum sativum L. var. Alaska) was examined using cell-free extracts. No tryptophan synthetase activity could be detected in the former. Using serine-14C and indole-14C, with extracts from peas, it is shown by chromatographic and radioautographic methods that tryptophan is the product of the reaction, that both the substrates are necessary, and that pyridoxal phosphate is a necessary cofactor. Inhibition by metal ions, lability, thermolability, and various other properties are described. Partial purification was effected by gel filtration. The enzyme activity in pea root extracts, as measured by indole utilization per unit fresh weight, is much lower than in the enzyme preparations from terminal buds. A system causing destruction of indole, other than tryptophan synthetase, is also present in crude root extracts.

Tryptophan synthetase in shoot and root tissue of pea seedlings

A method was developed for partial purification of the B-protein of tryptophan synthase (EC 4.2.1.20) from pea plants. The enzyme was purified 28-fold with about 21% recovery. The purification proc...

Tryptophan synthase: partial purification and some properties of the B-protein subunit from pea plants (Pisum sativum cv. Alaska)

An undialyzable, thermostable inhibitor of tryptophan synthase from pea plants, Escherichia coli, and Neurospora crassa occurs in the filtrate from acetone extraction of pea tissue. A procedure is described for purification of the inhibitor extracted from roots. The final, purified preparation is homogeneous as shown by disc electrophoresis on acrylamide gel. The molecular weight, estimated by gel filtration on Sephadex G-200, is about 18 000. The inhibitor is stable even on heating at pH 7.0 in a water bath at 100 °C and has a characteristic ultraviolet absorption spectrum. The inhibitor shows some protein-like properties and acid hydrolysates were found to contain substances sensitive to ninhydrin. However, the hydrolysates also contain substances, presumably sugars, sensitive to benzidine reagent, appreciable sugar in galactose equivalents, and some fluorescent substances. The inhibitor was also found in shoot tissue. In both root and shoot the inhibitor increases, on a protein basis, with age of the t...

Tryptophan synthase: purification, and some properties of an inhibitor from pea roots

Evidence is presented showing that tryptophan synthase (L-serine hydro-lyase (adding indole), EC. 4.2.1.20) from pea is composed of two components, A and B, analogous to the protein subunits reported for bacteria, algae, and tobacco enzyme. Separation of the two components was achieved by gel filtration on Sephadex G-100. In contrast with enzyme from tobacco the two components could not be separated by ammonium sulfate precipitation. However, the A component was removed by heating at 70 °C for 5 min. The B component, alone, catalyzed the condensation of L-serine plus indole to tryptophan (reaction 2) at a significant level. A combination of the A and B components was necessary for the formation of tryptophan from indole-glycerol phosphate (reaction 1); and conversion of indole-glycerol phosphate to indole (reaction 3). Reaction 3 could only be demonstrated with partially purified enzyme extract, and the reaction proceeded well in the absence of added pyridoxal phosphate but was increased by its presence. ...

Tryptophan synthase: a two-component enzyme from pea plants (Pisum sativum cv. Alaska)

Low tryptophan synthase (L-serine hydro-lyase (adding indole), EC 4.2.1.20) activity in extracts of pea roots is a consequence of both a low concentration of the enzyme in roots and the presence of inhibitors at least some of which may be removed by homogenizing the fresh tissue with acetone. Inhibition by root extracts, and fractions thereof, was assayed against tryptophan synthase from seedling buds. At least three inhibitory components were detected, namely (1) a dialyzable, thermostabile component, (2) an undialyzable, thermolabile component, and (3) an undialyzable, thermostabile component. A dialyzable factor which protects an undialyzable inhibitory factor against heat inactivation is also indicated.Fractionation of crude root extracts by gel filtration on a column of Sephadex G-50 shows inhibitor with a molecular weight of about 10 000 or larger.

W. G. Boll

Papers

Tryptophan synthetase in shoot and root tissue of pea seedlings

Tryptophan synthase: partial purification and some properties of the B-protein subunit from pea plants (Pisum sativum cv. Alaska)

Tryptophan synthase: purification, and some properties of an inhibitor from pea roots

Tryptophan synthase: a two-component enzyme from pea plants (Pisum sativum cv. Alaska)

Inhibition of tryptophan synthase by extracts from pea roots