Colin H. Doy

Development and differentiation of haploid Lycopersicon esculentum (tomato)

SummaryHaploid callus cultures of selected races of Lycopersicon (tomato) species can be obtained from anther culture. This is a further demonstration of a proposed general method of haploid culture developed with Arabidopsis thaliana. Differentiation of haploid callus of Lycopersicon esculentum can be controlled both in the dark and the light by hormones added to defined minimal media. Development to plantlets is achieved only in the light. Callus cells can be induced to develop into seedless pseudo-fruits. Chromosome counts on callus cells or root-tip cells establishes haploidy (n=12).Haploidy can be maintained in culture on defined minimal media for at least one year.

The separation of three allosterically inhibitable 3-deoxy-d-arabino-heptulosonate 7-phosphate synthases from extracts of Neurospora crassa and the purification of the tyrosine inhibitable isoenzyme

Abstract 1. 1. A method is described for the complete separation of 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy- d - arabino -heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) isoenzymes inhibited by tryptophan (DAHP synthase (Trp)), phenylalanine (DAHP synthase (Phe)) and tyrosine (DAHP synthase (Tyr)). 2. 2. DAHP synthase (Tyr) is purified to homogeneity as judged by electrophoresis on polyacrylamide gels. The presence of the substrate phosphoenolpyruvate is necessary for stability during purification and subsequent storage. 3. 3. The purification procedure makes use of allosteric ligand dependent reversible changes of molecular weight. Purification by ligand-dependent changes of molecular weight may have wider applications.

The nature of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in extracts of wild-type Neurospora crassa: A reaction controlled by two activating substrates and three allosteric negative modifiers

Abstract 1. 1. The 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy- d - arabino -heponate) d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase) of Neurospora crassa wild type 74A is a unit of physiological function but regultory differentiation. Kinetic measurements are made with crude extracts in order to minimise denaturation of possible unifying native molecular organisation. A number of tentative conclusions (see below) are reached regarding ligand-enzyme interactions. 2. 2. DAHP synthase is a thiol enzyme. Co 2+ stimulates variably, EDTA inhibits strongly and is reversed completely by Co 2+ . Between pH 6.2–8.0 there is little change from an optimal activity at about pH 6.5. An Arrhenius plot is linear without transition between 17° and 37°. Heat denaturation occurs above about 4 i °. ΔH ★ is approx. 16 000 cal/mole. Crude dialysed extracts are stable for at least 1 h at 37°, but are rapidly deactivated by dilution. 3. 3. The substrates erythrose 4-phosphate and phosphoenol pyruvate are also activators (positive modifiers) with strong cooperative interaction between a minimum of 2 active sites per substrate species ( m = 1.7–1.8). Apparent K m does not apply in these examples, but [ S ] 0·5 ( V ) for either substrate is about 1.8·10 −4 M. The reaction is presumptive ping-poing but includes cooperative site interactions. It is deduced that PEP adds first and the importance of PEP as an initiating metabolite of aromatic biosynthesis is discussed. 4. 4. At least three distinct kinds of active sites occur; those inhibited by phenylanine, those inhibited by tyrosine and those inhibited by tryptophan. The ratio between these is normally about 44:44:10. Inhibition by tryptophan has not previously been demonstrated at this site in Nature. These end products are negative modifers each showing strong cooperative interaction between at least two sites. They act in part by lowering cooperation between substrate molecules. One molecule of tyrosine may sometimes bind ( m = 0.9, [ M ] 0.5 = 6·10−6 M). The phenylalanine and tyrosine sites must partly interact since their inhibition pattern is synergistic. Inhibition by tyrosine and phenylalanine is noncompetitive with regard to each substrate. When m = 1.4 to 2, values of [ M ] 0.5 range from 8·10 −6 to 1.7·10 −5 for different negative modifiers. The enzyme is rapidly denatured at 45°. The phenylalanine-sensitive component is most labile, but selectively protected by phenylalanine. The tyrosine-sensitive component is selectively protected by phosphoenol pyruvate (PEP). The tryptophan-sensitive portion and a small non-inhibited portion survive for at least i h without protection. Heat denaturation does not create a non-inhibited portion.

Isoenzymes and allosteric inhibition of Neurospora crassa 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase

Abstract 1. 1. The molecular sieving on agarose of the 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy- d - arabino -heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase) from the fungus Neurospora crassa is described. Isoenzymes inhibited by the allosteric ligands, phenylalanine, tyrosine, and tryptophan (DAHP synthases (Phe), (Tyr) and (Trp)) were separated. The substrate phosphoenolpyruvate stabilizes all forms of DAHP synthases (Tyr) and (Phe) and is necessary for their recovery. 2. 2. The influence of all possible combinations of the three allosteric ligands on the apparent molecular weight of isoenzymes was determined in Tris-maleate and phosphate buffers (pH 7.4). It is concluded that DAHP synthases (Phe) and (Tyr) dissociate to half-molecules in the presence of their specific allosteric ligands but that DAHP synthase (Trp) does not. Non-inhibited activity was associated with the inhibited isoenzymes rather than existing independently. 3. 3.The non-inhibited DAHP synthases derived from DAHP synthases (Phe) and (Tyr) by mutation (allosteric inhibition-negative mutants) did not dissociate to half-molecules in the presence of the parental allosteric inhibitor. It is concluded that dissociation of the wild-type isoenzymes is part of the normal mechanism of allosteric inhibition. 4. 4. Non-exclusive binding of the substrate phosphoenolpyruvate and the allosteric ligands phenylalanine and tyrosine is proposed. 5. 5. Those features of the Neurospora crassa enzyme presently regarded as unique by comparison with Escherichia coli enzyme are summarized.

Studies concerning the biochemical genetics and physiology of activity and allosteric inhibition mutants of Neurospora crassa 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase

Abstract 1. 1. Procedures are described for the selection of two classes of presumptive structural gene mutants of the 3-deoxy- d -arabino-heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy- d -arabino-heptonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase) of the fungus Neurospora crassa. (a) Three separate activity-negative mutations have been correlated with isoenzymes inhibited by the end products, tyrosine, phenylalanine and tryptophan; arom-6 with DAHP synthase (Tyr), arom-7 with DAHP synthase (Phe) and arom-8 with DAHP synthase (Trp). (b) Mutants retaining activity but no longer allosterically inhibited (allosteric inhibition-negative) were obtained for each of the three isoenzymes. 2. 2. Arom-6, arom-7 and arom-8 are unlinked to the “arom gene cluster”. Arom-6 maps on linkage group VI L; arom-7 and arom-8 both map on linkage group I R but are widely separated. Therefore, no DAHP synthase operon or operon-like gene cluster exists. In all three cases, the absence of recombination and complementation in vitro (enzymic) and in vivo (forced heterocaryon) indicates that the two classes of mutations associated with a specific isoenzyme are allelic. On this hypothesis a single polypeptide prescribes activity and allosteric inhibition, but the polypeptide is different for each isoenzyme. 3. 3. Strains carrying arom-6 and grown in the presence of phenylalanine, tyrosine and tryptophan have a nutritional requirement for the folic acid precursor 4-aminobenzoate. This suggests that DAHP synthase (Tyr) plays a special regulatory role associated with the synthesis of 4-aminobenzoate. 4. 4. It is concluded that DAHP synthase is differentially controlled by phenylalanine, tyrosine and tryptophan without channelling of DAHP.

Transgenosis of Bacterial Genes from Escherichia Coli to cultures of Haploid Lycopersicon Esculentum and Haploid Arabidopsis Thaliana Plant Cells

What may be a general method for the production of haploid callus cultures of higher plants (angiosperms) on fully defined and minimal media has recently been developed in this laboratory (Gresshoff and Doy, 1972a, 1972b). Cell lines from pollen mother cells of the diploid plants Lycopersicon esculentum (tomato) and Arabidopsis thaliana have remained haploid throughout a year of sub-culture. Thus the genotypes of these plants are now carried by haploid cultures analogous to those of vegetatively dividing eukaryotic micro-organisms.

The exogenous environment as a factor in the control of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Neurospora crassa.

1. (1) The 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase activity (7 phospho-2-keto-3-deoxy-d-arabino-heptonated-erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase) found in wild type Neurospora crassa 74A grown for 60–66 h on minimal medium or in the presence of all possible combinations of aromatic amino acids, is the basal repressed level. 2. (2) Growth of an aromatic auxotroph on limiting aromatic amino acids results in at least a ro-fold derepression of the phenylalanine plus tyrosine inhibited portions of DAHP synthase. 3. (3) Addition of the aromatic amino acids to the growth medium is capable of inhibiting DAHP synthesis completely, or nearly completely. 4. (4) Methods have been developed for measuring DAHP or DAH in samples containing tryptophan and non-ionic inhibitors of the colorimetric assay.

Some properties of the 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase isoenzymes from mutant strains of Neurospora crassa

Abstract 1. 1. Extracts of mutant strains of Neurospora crassa which retain only one 3-deoxy- d -arabino- heptulosonate 7-phosphate synthase ( 7-phospho-2-keot-3-deoxy- d -arabino- heptulosonate d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating) EC 4.1.2.15) (DAHP synthase) isoenzyme have been used to test the properties of isoenzymes by direct analysis rather than by the differential inhibition analysis previously used for wild-type preparations. DAHP synthases (Phe) and (Tyr), in the presence of their specific negative effectors, show a 40% decrease in molecular weight as estimated by gel filtration. This is in accord with results obtained with wild-type preparations and is interpreted as indicating the dissociation of a polymer into subunits. DAHP synthase from mutants in which the remaining isoenzyme is insensitive to inhibition do not dissociate, implicating dissociation as an essential part of the normal inhibition mechanism. 2. 2. DAHP synthase (Trp) has not been found to dissociate in the presence of tryptophan. 3. 3. Evidence of an in vitro interaction between DAHP synthases (Phe) and (Tyr) has been obtained. The presence of a specific allele of the arom -6 locus (resulting in the loss of DAHP synthase (Tyr) activity) is correlated with a change in the gel-filtration characteristics of DAHP synthase (Phe), the product of an unlinked gene ( arom -7).