Mechanism of C-3 hydrogen exchange and the elimination of ammonia in the 3-methylaspartate ammonia-lyase reaction.

The enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) catalyzes the exchange of the C-3 hydrogen of the substrate, (2S,3S)-3-methylaspartic acid, with solvent hydrogen. The mechanism of the exchange reaction was probed using (2S,3S)-3-methylaspartic acid and its C-3-deuteriated isotopomer. Incubations conducted in tritiated water allowed the rate of protium or deuterium wash-out from the substrates to be measured as tritium wash-in. The primary deuterium isotope effects for the exchange under essentially Vmax conditions ( [S] much greater than Km) were 1.6, 1.5, and 1.5 at pH 9.0, 7.6, and 6.5. The deamination reaction, measured spectrophotometrically on the same incubations, showed isotope effects of 1.7, 1.6, and 1.4 at pH 9.0, 7.6, and 6.5, in agreement with the values of DV and D(V/K) reported previously [Botting, N.P., Akhtar, M., Cohen, M.A., & Gani, D. (1988) Biochemistry 27, 2956-2959]. The ratio of the rate of exchange to the rate of deamination, however, varied widely with pH. Together with the identical values of the primary isotope effects for the two reactions, this result indicates that the partition between reaction pathways occurs after the slowest steps in the common part of the reaction coordinate pathway, almost certainly after the cleavage of the C-N bond at the level of the enzyme-ammonia-mesaconic acid complex, and not at the putative carbanion level as was previously suggested. The enzyme requires both K+ and Mg2+ ions for activity, although ammonium ion is also able to bind in the K+ site and act as an activator. Variation of the metal ion concentration alters the magnitude of the primary deuterium isotope effects. The variation of potassium ion concentration causes the most marked changes: at 1.6 mM K+, DV and D(V/K) are 1.7, whereas at 50 mM K+, DV and D(V/K) are reduced to 1.0. The isotope effects are also reduced at low K+ concentration due to the emergence of a slow-acting high K+ affinity monopotassium form of the enzyme. The binding order and role of the metal ion cofactors and their influence in determining the formal mechanism of the reaction is discussed, and the failure of previous workers to observe primary deuterium isotope effects for the deamination process is explained. The product desorption order was tested by product inhibition, alternative product inhibition, and isotope exchange experiments. Ammonia and mesaconic acid debind in a random fashion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Quantitative trait locus analysis of a recombinant inbred line population derived from a Lycopersicon esculentum x Lycopersicon cheesmanii cross

Quantitative trait loci influencing fruit traits were identified by restriction fragment length polymorphism (RFLP) analysis in a population of recombinant inbred lines (RIL) derived from a cross of the cultivated tomato, Lycopersicon esculentum with a related wild species Lycopersicon cheesmanii. One hundred thirty-two polymorphic RFLP loci spaced throughout the tomato genome were scored for 97 F₈ RIL families. Fruit weight and soluble solids were measured in replicated trials during 1991 and 1992. Seed weight was measured in 1992. Significant (P<0.01 level) quantitative trait locus (QTL) associations of marker loci were identified for each trait. A total of 73 significant marker locus-trait associations were detected for the three traits measured. Fifty-three of these associations were for fruit weight and soluble solids, many of which involved marker loci signficantly associated with both traits. QTL with large effects on all three traits were detected on chromosome 6. Greater homozygosity at many loci in the RIL population as compared to F₂ populations and greater genomic coverage resulted in increased precision in the estimation of QTL effects, and large proportions of the total phenotypic variance were explained by marker class variation at significant marker loci for many traits. The RIL population was effective in detecting and discriminating among QTL for these traits previously identified in other investigations despite skewed segregation ratios at many marker loci. Large additive effects were measured at significant marker loci. Lower fruit weight, higher soluble solids, and lower seed weight were generally associated with RFLP alleles from theL. cheesmanii parent.     

Fern L-methionine decarboxylase: kinetics and mechanism of decarboxylation and abortive transamination

L-Methionine decarboxylase from Dryopteris filix-mas catalyzes the decarboxylation of L-methionine and a range of straight- and branched-chain L-amino acids to give the corresponding amine products. The deuterium solvent isotope effects for the decarboxylation of (2S)-methionine are D(V/K) = 6.5 and DV = 2.3, for (2S)-valine are D(V/K) = 1.9 and DV = 2.6, and for (2S)-leucine are D(V/K) = 2.5 and DV = 1.0 at pL 5.5. At pL 6.0 and above, where the value of kcat for all of the substrates is low, the solvent isotope effects on Vmax for methionine are 1.1-1.2 whereas the effects on V/K remain unchanged, indicating that the solvent-sensitive transition state occurs before the first irreversible step, carbon dioxide desorption. The enzyme also catalyzes an abortive decarboxylation-transamination reaction in which the coenzyme is converted to pyridoxamine phosphate [Stevenson, D. E., Akhtar, M., & Gani, D. (1990a) Biochemistry (first paper of three in this issue)]. At very high concentration, the product amine can promote transamination of the coenzyme. However, the reaction occurs infrequently and does not influence the partitioning between decarboxylation and substrate-mediated abortive transamination under steady-state turnover conditions. The partition ratio, normal catalytic versus abortive events, can be determined from the amount of substrate consumed by a known amount of enzyme at infinite time, and the rate of inactivation can be determined by measuring the decrease in enzyme activity with respect to time. For methionine, the values of Km as determined from double-reciprocal plots of concentration versus inactivation rate are the same as those calculated from initial catalytic (decarboxylation) rate data, indicating that a single common intermediate partitions between product formation and slow transamination. The partition ratio is sensitive to changes in pH and is also dependent upon the structure of the substrate; methionine causes less frequent inactivation than either valine or leucine. The pH dependence of the partition ratio with methionine as substrate is very similar to that for V/K. Both curves show a sharp increase at approximately pH 6.25, indicating that a catalytic group on the enzyme simultaneously suppresses the abortive reaction and enhances physiological reaction in its unprotonated state. Experiments conducted in deuterium oxide allowed the solvent isotope effects for the partition ratio and the abortive reaction to be determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ly-4.2: Doubts concerning its validity as a B-cell marker

The serum used to define Ly-4.2, (BALB/c × SWR/J)F₁ anti-B10D2/n, was found to react equally with both donor B and T cells, contrary to the findings of McKenzie and Snell (1975) that the serum reacted mainly with B cells. The reaction with both B and T cells was demonstrated by a 90 to 100 percent killing of spleen, lymph node, and separated populations of B and T cells from both organs. Although the antiserum only lysed 10 to 20 percent of thymocytes, these cells could be used to remove all reactivity against lymph node and spleen cells. Depletion of the T cells in a lymph node suspension by Thy-1.2 treatment did not affect the percentage of lysis of residual cells. Attempts to remove possible contaminating antibodies by absorption with either donor thymus or EL4 cells were unsuccessful because these tissues removed all antibody activity. Neither did partial absorption with separated B and T cells affect the relative activity against these cells. The strain distribution of Ly-4.2 was similar to that reported in the literature, and backcross tests indicated that there was probably no linkage with theH-2 locus.     

Efficacy of intercropping and harvest time modification for reducing field infestation of maize by Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)

Most management practices of Sitophilus zeamais Motschulsky, a field-to-post-harvest insect pest of cereals, have focused on post harvest control methods. This experiment was designed to investigate the potential of cropping system and modification of time of harvest to control S. zeamais. Intercropping and harvest time modification had significant (P < 0.05) effect on the number of S. zeamais emerging 42 days post-harvest. For the early harvest (15 weeks after planting (WAP)), the mean number of S. zeamais recorded from a maize monoculture (7.39) was significantly (P < 0.05) higher than the mean numbers of weevils emerging from a maize–soybean intercrop (2.31), but not significantly higher than the number recorded in maize–groundnut (3.87) intercrop. For the late harvest (18 WAP), the mean number of emerged adult observed in the maize–soybean intercrop (6.13) was significantly lower than the mean number of adult emerging from the monocrop maize (13.24). Maize–groundnut intercrop did not significantly reduce field infestation of S. zeamais compared with monocrop maize. Percentage weight loss observed in early harvested maize was significantly (P < 0.0001) lower than what was observed in late-harvested maize. Percentage weight loss was highest in stored maize harvested from monocrop maize plots for the early harvest, whereas intercropping maize with soybean reduced percentage weight loss when harvest was delayed.     

Fruit cuticle lipid composition and water loss in a diverse collection of pepper (Capsicum)

Pepper (Capsicum spp.) fruits are covered by a relatively thick coating of cuticle that limits fruit water loss, a trait previously associated with maintenance of postharvest fruit quality during commercial marketing. To shed light on the chemical‐compositional diversity of cuticles in pepper, the fruit cuticles from 50 diverse pepper genotypes from a world collection were screened for both wax and cutin monomer amount and composition. These same genotypes were also screened for fruit water loss rate and this was tested for associations with cuticle composition. Our results revealed an unexpectedly large amount of variation for the fruit cuticle lipids, with a more than 14‐fold range for total wax amounts and a more than 16‐fold range for cutin monomer amounts between the most extreme accessions. Within the major wax constituents fatty acids varied from 1 to 46%, primary alcohols from 2 to 19%, n‐alkanes from 13 to 74% and triterpenoids and sterols from 10 to 77%. Within the cutin monomers, total hexadecanoic acids ranged from 54 to 87%, total octadecanoic acids ranged from 10 to 38% and coumaric acids ranged from 0.2 to 8% of the total. We also observed considerable differences in water loss among the accessions, and unique correlations between water loss and cuticle constituents. The resources described here will be valuable for future studies of the physiological function of fruit cuticle, for the identification of genes and QTLs associated with fruit cuticle synthesis in pepper fruit, and as a starting point for breeding improved fruit quality in pepper.     

The A locus that controls anthocyanin accumulation in pepper encodes a MYB transcription factor homologous to Anthocyanin2 of Petunia

Pepper plants containing the dominant A gene accumulate anthocyanin pigments in the foliage, flower and immature fruit. We previously mapped A to pepper chromosome 10 in the F₂ progeny of a cross between 5226 (purple-fruited) and PI 159234 (green-fruited) to a region that corresponds, in tomato, to the location of Petunia anthocyanin 2 (An2), a regulator of anthocyanin biosynthesis. This suggested that A encodes a homologue of Petunia An2. Using the sequences of An2 and a corresponding tomato expressed sequence tag, we isolated a pepper cDNA orthologous to An2 that cosegregated with A. We subsequently determined the expression of A by Northern analysis, using RNA extracted from fruits, flowers and leaves of 5226 and PI 159234. In 5226, expression was detected in all stages of fruit development and in both flower and leaf. In contrast, A was not expressed in the sampled tissues in PI 159234. Genomic sequence comparison of A between green- and purple-fruited genotypes revealed no differences in the coding region, indicating that the lack of expression of A in the green genotypes can be attributed to variation in the promoter region. By analyzing the expression of the structural genes in the anthocyanin biosynthetic pathway in 5226 and PI 159234, it was determined that, similar to Petunia, the early genes in the pathway are regulated independently of A, while expression of the late genes is A-dependent.Communicated by R. Hagemann     

Genetic and molecular regulation of fruit and plant domestication traits in tomato and pepper

Tomato and pepper are two Solanaceous fruit crops that display an enormous diversity in fruit morphology. In this review, we will present an overview of the history of tomato and pepper and discuss key plant traits that were specifically selected during domestication of the two species. The traits discussed are fruit weight, shape, colour, ripening, pungency and plant architecture. We will review these characteristics as well as the genetic loci or genes that control these features, questioning whether mutations at orthologous loci occurred independently in these two species or whether unique plant and fruit features resulted in selection at different genes.