Segregation for endosperm lysine in F2, F3 and F4 progeny from a cross of in vitro-selected and unselected cultivar of rice

Summary Lysine is a limiting amino acid for optimal nutritional quality in rice grain. In vitro selections using inhibitory levels of lysine plus threonine or s-aminoethylcysteine allow the predictable recovery of variants with elevated levels of lysine and protein. These methods may generate useful starting germplasm for plant breeders. This study was conducted to define the genetics of lysine mutants in progeny from crosses of mutants derived from cells cultured in vitro in the presence of inhibitory levels of lysine plus threonine and s-(2-aminoethyl)-cysteine. In vitro selections produce a wide range of mutants, including endosperm mutants with elevated lysine and protein levels as well as mutants for high and low seed weights. Mutants were analyzed for lysine content by the endosperm half-seed method in which the halves without the embryo were ground and acid hydrolyzed for amino acid determinations. The halves with the embryos were preserved for later germination. In two different F₂ populations derived from a cross of a selected mutant x M-101, a parental marker, there was an inverse relationship between seed weight and percent lysine in endosperm protein (R² 0.52 and 0.56). The F₂ segregation patterns show that elevated lysine is inherited as a recessive gene and that increased lysine is correlated with decreased seed size. F₃ and F₄ data provide evidence for the transmission of high lysine genes to advanced germplasm in rice. This work supports our earlier conclusions that high lysine phenotypes can be recovered predictably from in vitro selections. The elevated lysine phenotypes are frequently, but not exclusively, associated with opaque seed. Some segregants from crosses produced increased lysine in plants with near normal seed weight and good fertility.Research done under the auspices of the USDA, ARS, Plant Sciences Institute, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA     

Electrophoretic profiles and amino acid composition of rice endosperm proteins of a mutant with enhanced lysine and total protein after backcrosses for germplasm improvements

 Seed storage proteins from in vitro-derived rice mutants improved by several backcrosses to ‘Calrose 76’ and BC₂ and BC₃ were characterized for changes in five different solubility classes. Albumins, rsealb (water-soluble globulins), true salt-soluble globulins, prolamins and glutelins were SDS-PAGE separated in a single dimension, and some two-dimensionally, to identify protein modifications. The genetic transmission of the enhanced-lysine mutants in backcrosses and the linkage of lysine with grain chalkiness were confirmed. Advanced lines had altered globulin profiles similar to those of unimproved lines. Chalky/ enhanced-lysine phenotypes had similar prolamin and glutelin profiles in the mutant and controls at the same protein level. Mutants had increased levels of globulins at 50 kDa and 33 kDa but had substantially less protein at 25 kDa than the controls. High protein in the mutant contributed to an increase in prolamins and the major storage proteins in both the globulins and glutelins. A significant decrease in low-molecular-weight, 15- to 18-kDa albumins was associated with the chalky/enhanced-lysine mutant phenotype. Two proteins in the 15- to 18-kDa group were amino acid sequenced, and database comparisons identified these proteins as allergens. Advanced lines downregulated for allergens and with enhanced-lysine/protein but with normal fertility and seed weight should be useful in breeding programs for nutritional quality. Received: 14 October 1996 / Accepted: 8 November 1996     

Rice protein mutant expressed in liquid suspension cultures: chitinases, β-glucanases and other proteins

Summary A rice mutant with unique protein expression/ transport properties has been established as cells in liquid suspension and partially characterized. Mutants were originally recovered from anther calli grown for three cycles at inhibitory levels of lysine + threonine and one cycle of S-(2-aminoethyl)cysteine. Cell suspension cultures were started from high lysine-containing seeds regenerated from the inhibitor selections. Cultures of the mutant produce 2 times as much protein per unit weight as is produced by the control. Significant portions of the proteins are exported from the cells into the surrounding medium. The mutant also has 20% greater lysine content in the exported protein than the control. This cell suspension line should be particularly useful for biochemical and molecular studies on protein synthesis and processing phenomena in cereals.Research done under the auspices of the USDA, ARS, Plant Science Institute, Plant Molecular Biology Laboratory, Beltsville, Md 20705, USA     

Free and bound amino acids and proteins in developing grains of rice with enhanced lysine/proteins

 Free amino acids were determined in developing seed of a rice mutant with enhanced grain lysine. This phenotype frequently has enhanced protein. Some free amino acids of developing seed are inversely related to the level of total amino acids in proteins of the mature grain. Amino acids that were enhanced in protein, including aspartic acid, threonine, methionine and lysine, were notably lower in the free amino-acid pool. Our conclusion is that mutant-developing grains process aspartate amino acids more rapidly than the controls. Conversely, arginine, valine and glutamic acid/glutamine accumulate as free amino acids with mutant/control ratios of 1.39, 1.29 and 1.12, respectively. Glutamic acid/glutamine in proteins of mature seeds is lower in the mutant than the control. ³H-lysine incorporation showed enhanced isotope incorporation into at least four proteins. One mutant protein was less actively labelled than analogous controls. The ³Hlysine pattern indicates processing modifications in this useful rice mutant. Received: 14 October 1996/Accepted: 8 November 1996     

Increased Lysine and Seed Storage Protein in Rice Plants Recovered from Calli Selected with Inhibitory Levels of Lysine plus Threonine and S-(2-Aminoethyl)cysteine

Experiments were designed to test whether variation in percent lysine in seed proteins could be recovered in plants regenerated from callus subjected to inhibitory levels of lysine plus threonine. Anther-derived callus was subjected to 1 millimolar lysine plus threonine for three successive passages and then once to the same concentration of S-(2-aminoethyl)cysteine. Plants were regenerated from the resistant callus. Plants recovered directly from tissue culture were normal in color, size and were 50% or less fertile. Second and third generation plants produced a wide range of variants including albinos, deep green plants both short and tall, and totally fertile as well as partially fertile plants. All regenerated plants produced chalky or opaque seed. One unique second generation line had 14% more lysine in seed storage proteins than the controls. This characteristic was transmitted to the next generation. The high lysine plants had reduced seed size with significantly higher levels of seed storage protein than the controls. The phenotypes recovered provide experimental materials for basic studies in protein synthesis and lysine metabolism and may become a source of material for rice breeding.     

Opaque7 encodes an acyl-activating enzyme-like protein that affects storage protein synthesis in maize endosperm

In maize, a series of seed mutants with starchy endosperm could increase the lysine content by decreased amount of zeins, the main storage proteins in endosperm. Cloning and characterization of these mutants could reveal regulatory mechanisms for zeins accumulation in maize endosperm. Opaque7 (o7) is a classic maize starchy endosperm mutant with large effects on zeins accumulation and high lysine content. In this study, the O7 gene was cloned by map-based cloning and confirmed by transgenic functional complementation and RNAi. The o7-ref allele has a 12-bp in-frame deletion. The four-amino-acid deletion caused low accumulation of o7 protein in vivo. The O7 gene encodes an acyl-activating enzyme with high similarity to AAE3. The opaque phenotype of the o7 mutant was produced by the reduction of protein body size and number caused by a decrease in the α-zeins concentrations. Analysis of amino acids and metabolites suggested that the O7 gene might affect amino acid biosynthesis by affecting α-ketoglutaric acid and oxaloacetic acid. Transgenic rice seeds containing RNAi constructs targeting the rice ortholog of maize O7 also produced lower amounts of seed proteins and displayed an opaque endosperm phenotype, indicating a conserved biological function of O7 in cereal crops. The cloning of O7 revealed a novel regulatory mechanism for storage protein synthesis and highlighted an effective target for the genetic manipulation of storage protein contents in cereal seeds.     

Screening of Lysine-rich Plant Species and Identification of the Purified Proteins

The nutritional quality of seed proteins from cereals, such as wheat and rice, is comparatively low due to its deficency in lysine and some essential amino acids. In this research extensive varieties of plant seed samples were collected and screened by analysis of amino acid composition. Three lysing-rich species which contain more than 6.7% of lysine in total seed proteins were found. 31 kinds of proteins were purified from a species which contains 7.9% of lysine using the modified methods of IEF and SDS electrophoresis. One protein with PI 6.1 and 18 kD was identified which contains 11.4% of lysine and was rich in threonine, valine and isoleucine. This is the first example of the protein which could complement several limiting amino acids of wheat or rice. Further research on the structural gene encoding this protein would have great potential value for improvement of protein quality of these cereals.     

The screening and preliminary construction of quality mutant population for cultivar “Nipponbare” in japonica rice (Oryza sativa)

Progenies of Oryza sativa cv. Nipponbare induced with 0.4% ethyl methane sulphonate (EMS) were screened for quality mutants and the preliminary quality mutant population was constructed in present experiment. A total of 2210 materials were first screened using near infrared reflectance spectroscopy (NIRS) from which 208 quality mutants were obtained for a second screening and then yielded 73 quality mutants including amylase content (AC), gel consistency (GC), gelatinization temperature (GT), protein content (PC), rapid viscosity analysis (RVA) parameters and amino acid contents. The screening yielded 11 PC mutants with a mutation frequency of 4.98??, followed by 7 rice floury viscosity mutants (3.17??), 5 AC mutants (2.26??), 4 chalky mutants, GT and GC mutants (1.81??), and 2 ASV mutants (0.9??). The relative contents of 17 kinds of amino acid mutations, including 7 kinds for essential amino acids and 10 kinds for nonessential amino acids were identified. With the variation of 10% as the screening standard, mutants were obtained for lysine and leucine at 0.45?? and for valine at 4.98??, but no mutants were found for isoleucine, phenylalanine, threonine. For nonessential amino acids, mutants of glutamic (0.45??), arginine (3.62??), alanine (3.17??), serine (0.45??), glycine (0.45??), tyrosine (1.81??), proline (2.71??), and histidine (0.45??) were obtained, but none was found for aspartic, phenylalanine nor threonine. At 100% as the screening standard for methionine and cysteines, the mutation frequency of these two amino acid mutants were 0.9?? and 4.98?? respectively. Quality mutants in this preliminary library of rice could play important role in gene function and breeding of rice quality.     

A rice homolog of Cdk7/MO15 phosphorylates both cyclin-dependent protein kinases and the carboxy-terminal domain of RNA polymerase II

The activation of cyclin-dependent protein kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop by a CDK-activating kinase (CAK). The R2 protein of rice is very similar to CAKs of animals and fission yeast at the amino acid level but phosphorylation by R2 has not yet been demonstrated. When R2 was overexpressed in a CAK-deficient mutant of budding yeast, it suppressed the temperature sensitivity of the mutation. Immunoprecipitates of rice proteins with the anti-R2 antibody phosphorylated human CDK2, one of the rice CDKs (Cdc2Os1), and the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II of Arabidopsis. Mutational analysis indicated that R2 phosphorylated the threonine residue within the T-loop of CDK2 and Cdc2Os1. R2 was found mainly in two protein complexes which had molecular masses of 190 kDa and 70 kDa, respectively, whilst the CDK- and CTD-kinase activities associated with R2 were identified in a complex of 105 kDa. These results indicate that R2 is closely related to CAKs of animals and fission yeast in terms of its phosphorylation activity and, moreover, that this CAK of rice is distinct from a CAK of the dicotyledonous plant Arabidopsis.     

Fertility,Inheritance and Amino Acid Analysis of Lysine Plus Threonine-Resistant Mutant Progenies of Maize

Sixth generation of mutant maize seed homozygous for lysine plus threonine resistancewhich was derived from the resistant callus cultures has been harvested. The resistance could be inherited stably. The fertility, however, was very poor. The resistant homozygotes have been obtained by backcross of the wild type with the resistant plants (W77-R3019 ×R0), and their fertility could be parlty recovered after selection for the resistant plants from backcross progenies. Genetic analysis showed that the resistance inherited as a single dominant nuclear allele. All of the free amino acids except phenylalan inc in the homozygote are increased by 4 folds. and free essential amino acids by 5 folds which are higher than those in the wild types. Total amino acids increased by 5.53%. The dramatic increase (11 times) in free threonine adds up the total threonine by 17.73%. Difference of the protein content between the homozygote and wild type was not obvious. These results show that selection for the resistance to lysine plus threonine in maize and other cereals is probably very useful for improving their value of protein nutrition.     

Regulatory mutants of Streptomyces clavuligerus affected in free diaminopimelic acid content and antibiotic biosynthesis

The composition of intracellular free amino acid pools was determined in Streptomyces clavuligerus mutants possessing an altered aspartokinase which is insensitive to concerted feedback inhibition by threonine and lysine. These mutants contained total free amino acid pool contents that were considerably higher than those found in the wild-type strain. Diaminopimelic acid accounted for 10 to 20% of the total free amino acid pools, depending on the individual mutant and its culture growth phase, whereas diaminopimelic acid contained in the wild-type strain accounted for only 0.5% of the total free amino acid pool.     

Effect of Pyruvate Kinase Deficiency on l-Lysine Productivities of Mutants with Feedback-resistant Aspartokinases

The cultural conditions were investigated for a Brevibacterium flavum mutant, No. 2–190, with a low level of citrate synthase (CS) and with feedback-resistant phosphoenoipyruvate (PEP) carboxylase and aspartokinase (AK). The productivity was increased from 28 to 38 g/1 (as the HC1 salt) with a medium containing 10% glucose. From this strain, pyruvate kinase (PK)-defective mutants were derived and selected as to the inability to grow on ribose. Among them, strain Kl-18 showed higher lysine productivity than the parent under all cultural conditions tested, and produced 43 g/1 of lysine, at maximum. A lysine-producing mutant, No. 536–4, with a feedback- resistant AK was derived from PK-defective strain KH-21 which had low CS activity and a feedback-resistant PEP carboxylase. The mutant was isolated by a new selection method, that is, on the basis of resistance to α-amino-ß-hydroxyvaleric acid, a threonine analogue plus lysine. In this strain, HD had been altered so as to become feedback-resistant at the same time, resulting in the byproduction of threonine and isoleucine. The total amount of these aspartate family amino acids was higher on molar basis than that of lysine produced by strain No. 2–190.     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

Genetic and amino-acid analysis of two maize threonine-overproducing,lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37–54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.     

Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.     

Size Distribution and Amino Acid Chemistry of Base-extractable Proteins from Washington Coast Sediments

Proteinaceous components from four Washington coast margin sediments were extracted with base, fractionated into one of four size classes (<3 kDa, 3–10 kDa, 10–100 kDa, >100 kDa), and analyzed for their amino acid contents. Base-extracted material accounts for ~30% of the total hydrolyzable amino acids (THAA) and each size fraction has a unique composition, regardless of where the sediment was collected (shelf or upper slope). The <3 kDa size fraction (~10% of base-extractable THAA) is relatively enriched in glycine (~30 mol%), lysine (~5 mol%), and non-protein amino acids (~5 mol%). Glycine and non-protein amino acids are common degradation products, and lysine is very surface active. We suggest that the <3 kDa size fraction, therefore, represents a diagenetic mixture of fragments produced during the degradation of larger proteins. The 3–10 and 10–100 kDa size fractions (~10% and 42% of base-extractable THAA, respectively) have similar amino acid distributions dominated by aspartic acid (~30 mol%). Enrichments in Asp is likely due to both preservation of Asp-rich proteins and the production of Asp during degradation. The >100 kDa size fraction (~38% of base-extractable THAA) is not dominated by any particular amino acid and can not be modeled by mixing the amino acid compositions of the other size fractions. We propose that the larger size fractions (10–100 kDa and >100 kDa) represent intact, or near intact, proteins. Estimates of isoelectric points and relative hydrophobicity suggest the base-extractable proteins are primarily acidic and have globular structures. Statistical comparisons to several known proteins indicates that the base-extractable component is most similar to planktonic cytoplasmic proteins.     

Mutants for rice storage proteins

Summary To obtain genetic materials to breed qualitatively improved rice storage proteins, we screened about 3,000 mutant lines induced by the treatment of rice fertilized egg cell with N-methyl-N-nitrosourea (MNU). The screening was performed by comparing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with that of the original variety, Kinmaze, especially focussing on the changes in polypeptides present in two kinds of protein bodies, PB-I and PB-II. We selected 17 mutant lines and classified them into 4 types on the basis of variations of the relative contents of the polypeptides. Determination of extracted protein in the starchy endosperm of the mutants revealed changes in the content of prolamin and glutelin but not globulin. In some mutants there was marked accumulation of 57 kDa polypeptide concomitant with the remarkable reduction of glutelin subunits. Treatment of the fertilized egg cell with MNU was found to be an effective method to induce mutations for storage proteins in protein bodies of rice.     

Proteome analysis of programmed cell death and defense signaling using the rice lesion mimic mutant cdr2

We have previously identified three lesion-mimic mutants, cell death and resistance (cdr), in rice. These mutants induce a series of defense responses, including expression of defense-related genes and high accumulation of phytoalexins, indicating that the cdr mutants are useful materials to study programmed cell death and defense signaling in rice. Here, we carried out a proteome analysis of the cdr2 mutant. Total proteins prepared from the wild type and the cdr2 mutant at three different stages of lesion formation were compared using two-dimensional electrophoresis. We found a total of 37 proteins that were differentially expressed between cdr2 and wild type. Among them, 28 spots were up-regulated and nine were down-regulated in the cdr2 mutant. All the protein spots were identified by mass spectrometric analysis. These differentially regulated proteins included defense-related proteins. In addition, 27 proteins were classified as metabolic enzymes, suggesting that the programmed cell death that occurs in the cdr2 mutant is associated with active metabolic changes. Our study shows that proteome analysis is a useful approach to study programmed cell death and defense signaling in plants.     

Characterization of wild-type and mutant M13 gene V proteins by means of 1H-NMR

Recording of good quality NMR spectra of the single-stranded DNA binding protein gene V of the bacteriophage M13 is hindered by a specific protein aggregation effect. Conditions are described for which NMR spectra of the protein can best be recorded. The aromatic part of the spectrum has been reinvestigated by means of two-dimensional total correlation spectroscopy. Sequence-specific assignments were obtained for all of the aromatic amino acid residues with the help of a series of single-site mutant proteins. The solution properties of the mutants of the aromatic amino acid residues have been fully investigated. It has been shown that, for these proteins, either none or only local changes occur compared to the wild-type molecule. Spin-labeled oligonucleotide-binding studies of wild-type and mutant gene V proteins indicate that tyrosine 26 and phenylalanine 73 are the only aromatic residues involved in binding to short stretches of single-stranded DNA. The degree of aggregation of wild-type gene V protein is dependent on both the total protein and salt concentration. The data obtained suggest the occurrence of specific protein-protein interactions between dimeric gene V protein molecules in which the tyrosine residue at position 41 is involved. This hypothesis is further strengthened by the observation that the solubility of tyrosine 41 mutants of gene V protein is significantly higher than that of the wild-type protein. The discovery of the so-called 'solubility' mutants of M13 gene V protein has finally made it possible to study the solution structure of gene V protein and its interaction with single-stranded DNA by means of two-dimensional NMR.