Content uploaded by Steffen Strobaek
Author content
All content in this area was uploaded by Steffen Strobaek on Mar 22, 2016
Content may be subject to copyright.
A preview of the PDF is not available
On the nature of the polymorphism of the small subunit of ribulose-1,5-diphosphate carboxylase in the amphidiploid Nicotiana tabacum
Abstract
The N-terminal amino acid sequence of the small subunit of ribulose-1,5=disphosphate carboxylase from the amphidiploidNicotiana tabacum contains two polymorphisms. From examination of the equivalent sequences in the putative parent speciesNicotiana sylvestris andtomentosiformis it is concluded that the amphidiploidNicotiana tabacum has inherited two alleles for the small subunit of ribulose diphosphate carboxylase, one from each parent species. The alleles
continue to be retained and expressed. The relevance of these findings is discussed in relation to the successful adaption
of the amphidiploidNicotiana tabacum to a wide range of environments.
... Nicotiana tabacum which is likely to be the result of a fusion of Nicotiana sylvestris and Nicotiana tomentosiformis (Strobaek et al. 1976). This type of duplication however, can give rise only to dispersed multi-gene families. ...
Emil Heitz (1928) discovered that the chromocentres of the interphase nucleus constitute heterochromatic portions of chromosomes as contrasted to their euchromatic parts. His is the introduction of the terms heterochromatin and euchromatin as well as the recognition of different categories of heterochromatin. In spite of the large effort by cytologists and biochemists to elucidate the features of this chromatin differentiation a notion of Heitz in 1955 remains essentially true today. No definitive answer has been obtained as to the nature of the structural difference between eu- and heterochromatin nor is the genetic and functional significance of heterochromatin clear. Unfortunately, up to this day heterochromatin is frequently used as a scape-goat.
Biochemical studies on leaf proteins carried out by Wildman and Bonner (1947) revealed the presence of a major protein component having a large molecular size (18s) which was designated as fraction-1-protein. The ubiquitous distribution of this protein in green plant leaves and green algae, as determined by analytical ultracentrifugation and immunological precipitation methods, stimulated later studies on its enzymic nature (Dorner et al., 1958). The independent investigation of the path of carbon in photosynthetic CO2 fixation, together with that on the enzymic machinery of the reductive pentose phosphate cycle, led to the discovery of ribulose-1,5-bisphosphate (RuBP) carboxylase (E.C. 4.1.1.39; carboxydismutase) catalyzing the following key reaction [Eq. (1); Quayle et al., 1954; Weissbach et al., 1954, 1956; Chap. II.1, this vol.] $$
{\rm{RuBP + C}}{{\rm{O}}_2}{\rm{ }}{\rm{ 2}}\left( {{\rm{PGA}}} \right)
$$ (1)
There are scarcely any characters in systematics that have generated more controversy during their long period of application than that of the proteins. On the one hand their high a priori significance tempts an experimentator to extend, often inadmissibly, the a posteriori significance of his results. While, on the other hand, exaggerated criticism may arise when results from comparative protein experiments are at odds with the current concepts of relationships. An example of this comes from amino acid sequencing, where initial reflections (BOULTER et al., 1970) have led to a too optimistic view, some a posteriori considerations (CRONQUIST, 1976), however, to an underestimation of its significance in systematics.
Studies on the amino acid sequences and three-dimensional structures of plant proteins have always lagged far behind those of proteins from other sources. Thus in 1969, only six complete sequences for plant proteins had been established compared to over 230 from other sources (see Dayhoff 1972). At that time the tertiary structure of only one plant protein had been established with a reasonable degree of certainty (Drenth et al. 1968). The major reason for this apparent lack of interest in plant proteins probably lies in the relative difficulty in obtaining sufficient quantities of material for study from plants when compared to other sources. This is due to both the lower intrinsic yields of the plant proteins and to specific difficulties in their preparation. Thus even a decade later plant enzymes have still been barely studied because the conservative nature of many of the biochemical pathways means that similar enzymes are more readily available elsewhere. Even when there are enzymes unique to plants, such as photosynthetic enzymes, these have not been examined.
This chapter discusses the intergenomic interaction, heterosis, and improvement of crop yield. The genetic hierarchy in the multigenomic cell as a complex of all cell genetic determinants is distributed into chromosomal or nuclear genome and extrachromosomal plasmon, comprising the cytoplasmic organelles—mitochondria and chloroplasts. Both mitochondria and chloroplasts are self-replicating organelles, arising only by growth and division of pre-existing mitochondria and chloroplasts. The organelle genetic systems are thus genetically autonomous, obeying their own unique laws of uniparental or biparental inheritance. The population of organelle genomes within a cell is divided into subpopulations in individual organelles and in nucleoids. These “populations within populations” complicate the analysis of organelle transmission genetics. Organelle heterosis and complementation are significantly correlated with grain yield heterosis in many crops. The use of mitochondrial complementation or chloroplast complementation as a tool to pre-select well-combining parents under laboratory conditions for prediction of grain yield heterosis in crop plants is recommended. The improvement of crop yield in terms of mitochondrial and chloroplast breeding, especially in cereal crops, is a real challenge to modern plant geneticists, and the maturity of the field is suggestive of the fact that complementation data of organelles in 1:1 parental mixture should be preferred over time-consuming and expensive biometrical methods and estimations, because apparently both plasmon and nuclear genes are involved.
The endo-type glucanohydrolases ß-1,3-glucanase (E.C. 3.2.1.39) and chitinase (E.C. 3.2.1.14) are abundant proteins widely distributed in seedplant species (Clarke and Stone, 1962; Ballance and Manners, 1978; Powning and Irzykiewicz, 1965). The physiological functions of ß-1,3- glucanase and chitinase are not known. Based on the distribution of the enzyme and its putative substrates such as callose, it has been proposed that ß-1,3-glucanases may have a role in fruit ripening (Hinton and Pressey, 1980), pollen tube growth (Roggen and Stanley, 1969; Ori et al., 1990), coleoptile growth (Masuda and Wada, 1967), regulation of transport through vascular tissues (Clarke and Stone, 1962), cellulose biosynthesis (Meier et al., 1981) and cell division (Waterkeyn, 1967; Fulcher et al., 1976). Although the existence of other substrates has not been ruled out, chitin, the known substrate of chitinase, is not found in higher plants.
In previous papers we have reported the N-terminal 40 amino acids of the small subunit of rubisco for samples from four families of gymnosperms, nine families of monocotyledons, and 26 families of dicotyledons. We expanded this list to 122 families of dicots and derived a phylogenetic tree for all 335 species. The main computing program used was HENNIG86, with which a reliable result can be assured with only 17 taxa or less, so a major part of this paper is concerned with the strategy adopted to divide the 335 species and then to build the parts into an overall tree that is as accurate and objective as possible. Comparison with other taxonomy suggests that, at the level of placing genera into families, our methods give results that are at least 90% accurate. At higher taxonomic levels accuracy may decrease, and the result should be regarded not as a firm conclusion but as a working hypothesis for subsequent testing using the longer sequences from nucleic acids. Topics discussed include heterogeneity within species, the nature of the N-terminus of rubisco-SSU, and evidence that natural selection is powerful in determining amino acid sequence. The rate of evolution has been shown to vary between major taxa, and data suggest that angiosperms originated in the Jurassic.
The data on the primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase are reviewed. Examples of their use as markers and in the elucidation of the evolution, adaptation and function of this key enzyme are given.
This chapter reviews the reduction and S-carboxymethylation of proteins. Of the reagents available for the reduction of disulfide bonds, one of the most satisfactory is β-mercaptoethanol. The reduction in the absence of oxygen is performed with a large excess of reagent at a slightly alkaline pH and under conditions in which the protein is unfolded. Concentrated urea solutions, 8 or 10 M, have proved suitable as solvents. The carboxymethylation reaction generates iodide ions. To prevent the light-catalyzed oxidation of iodide to iodine, it is essential to perform the carboxymethylation step and the subsequent removal of reagents such as salts in the dark. Reduced carboxymethylated proteins may be isolated by procedures similar to those used for the reduced proteins. Many reduced carboxymethylated proteins are insoluble in water and can be obtained from the reaction mixture by dialysis. The extent to which complete reduction and S-carboxymethylation have been attained is determined by amino acid analysis. This also serves as a check on the extent of side reactions such as sulfonium salt or sulfoxide formation from methionine and modification of histidine or lysine. The chapter also discusses the reduction and S-carboxymethylation of ribonuclease A. Howevere, S-carboxymethylcysteine is determined with an amino acid analyzer.
Carboxypeptidases are enzymes that remove amino acids one residue at a time from the C-termini of polypeptide chains. Three types of enzyme are characterized that differ in the rate at which they release particular amino acids. This chapter discusses the enzymic hydrolysis with carboxypeptidases. Carboxypeptidases A (CPA) release most rapidly amino acids with an aromatic or large aliphatic side chain, hile carboxypeptidases B (CPB) release the basic amino acids lysine and arginine much faster than any of the other common protein amino acids. Plants contain carboxypeptidase C that liberates the amino acid proline as well as being able to release many of the other protein amino acids. CPA is generally prepared from ox pancreas by Anson's method. After recrystallization the material is contaminated with other pancreatic proteases, and must be treated with diisopropyl phosphorofluoridate (DFP) before being usable. The methods used in the preparation of enzyme solutions and protein substrate are described. Before a quantitative determination of the rate of release of amino acids from a protein is attempted, a preliminary experiment is carried to determine whether reaction takes place. The rate of hydrolysis of a peptide bond by a carboxypeptidase is affected by the nature of both the residues forming it, but the effect of the amino acid that is released predominates. Methods connected with the use of carboxypeptidase are hydrazinolysis, digestion in water, and modification of proteins by treatment with carboxypeptidases.
Using polyacrylamide gel slab electrophoresis, eight enzyme systems in leaf extracts of N. sylvestris, N. tomentosiformis, N. otophora, their interspecific hybrids, N. tabacum var. Burley 21 and Hicks, and Kostoff's amphidiploid 4X (N. sylvestris x N. tomentosiformis) have been compared in an attempt to elucidate their phylogenetic relationships. The degree of enzyme polymorphism appeared to be extensive for peroxidase and esterase, but was less so for chorogenic acid oxidase, catalase, acid phosphatase, malate dehydrogenase, leucine aminopeptidase, and glucose-6-phosphate dehydrogenase. Interspecific variations were evident for all enzyme systems analyzed. Zymograms from F1 plants exhibited a pattern that was an addition of those of the parent species, suggesting that these enzymes were controlled by dominant genes or codominant alleles in chromosomes of different genomes. An exception was that malate dehydrogenase isozymes in F1 hybrids tended to resemble the female parent in banding intensity. With reference to the relative mobility of isozymes for eight enzyme systems, the phylogenetic affinities in terms of similarity index values (s values) of N. tabacum with the F1 N. sylvestris x N. otophora and the N. sylvestris x N. tomentosiformis hybrid were 64% and 86% respectively. This supports the hypothesis that ancestors of N. sylvestris and N. tomentosiformis were likely the progenitors of N. tabacum. The Kostoff's amphidiploid showed the same zymograms as the F1 N. sylvestris x N. tomentosiformis for catalase, leucine aminopeptidase, and glucose-6-phosphate dehydrogenase; but possesses peroxidase, acid phosphatase, and malate dehydrogenase isozymes identical in mobility to those of N. tabacum. The s value between N. tabacum and this amphidiploid reached as high as 92%. The present results and other morphological and genetic evidence point to the possibility of introgression with N. tabacum when this amphidiploid was produced or in later stages of seed increase.
A simple, rapid, and reliable technique for identifying small amounts of phenylthiohydantoin (PTH) derivatives has been developed for use in the analysis by Edman degradation of small amounts of peptides isolated from fingerprints (10). Solvent systems used in paper and column chromatography of PTH amino acids have been adapted for use in thin-layer chromatography (TLC) on precoated flexible TL sheets with an incorporated fluorescent indicator (7). Twenty PTH amino acids can be identified on one TL sheet by ascending chromatography in one direction by the repeated utilization of two different solvent systems.