Effects of Nuclear and Cytoplasmic Genes Altering Chlorophyll Loss on Gas Exchange during Monocarpic Senescence in Soybean

Several genes can alter the yellowing processes that normallyoccur during pod development and monocarpic senescence in soybean.CytG and d₁ + d₂ cause the leaves and seeds to stay green atmaturity. G blocks yellowing of the seed coat but not the leavesor embryos. By contrast, another gene, y₃, causes earlier yellowingof the leaves. This paper examines the effects of these geneson photosynthesis and some related parameters of the senescencesyndrome in near-isogenic backgrounds (cv. Clark). Neither cytGnor d₁ d₂ delays the decline in photosynthetic rate during monocarpicsenescence relative to Clark; while Gd₁ d₂ does. Again, y₃ causesan earlier decrease. Similarly, neither cytG nor d₁ d₂ altersthe decline in stomatal conductance and transpiration, whichoccurs at the end of podfill; however, Gd₁ d₂ delays it andy₃ advances it. Neither cytG nor d₁ d₂ prevents the rise inintercellular CO₂ during podfill, while Gd₁ d₂ does, and y₃promotesit. These changes in intercellular CO₂ may reciprocally reflectthe status (maintenance for Gd₁ d₂ and early loss for y₃) ofthe photosynthetic enzymes. Gd₁ d₂, d₁ d₂ and cytG do not appreciablyaffect leaf blade abscission; however, y₃ prevents it. Gd₁ d₂increases the dry weight seed yield, and y₃ decreases it. Gd₁d₂ inhibits several components in the senescence process, whilecytG seems to affect mainly chlorophyll loss and y₃ may actmore broadly than just accelerating senescence. Clearly, thestay-green trait can be caused by more than one genetic locus,and it may or may not maintain photosynthesis. ³Present address: Instituto de Fisologia Vegetal, Facultad deAgronomia, c.c. 31, 1900-La Plata, Argentina (Received May 18, 1990; Accepted September 4, 1990)     

Active Degradation Explains the Distribution of Nuclear Proteins during Cellular Senescence

The amount of cellular proteins is a crucial parameter that is known to vary between cells as a function of the replicative passages, and can be important during physiological aging. The process of protein degradation is known to be performed by a series of enzymatic reactions, ranging from an initial step of protein ubiquitination to their final fragmentation by the proteasome. In this paper we propose a stochastic dynamical model of nuclear proteins concentration resulting from a balance between a constant production of proteins and their degradation by a cooperative enzymatic reaction. The predictions of this model are compared with experimental data obtained by fluorescence measurements of the amount of nuclear proteins in murine tail fibroblast (MTF) undergoing cellular senescence. Our model provides a three-parameter stationary distribution that is in good agreement with the experimental data even during the transition to the senescent state, where the nuclear protein concentration changes abruptly. The estimation of three parameters (cooperativity, saturation threshold, and maximal velocity of the reaction), and their evolution during replicative passages shows that only the maximal velocity varies significantly. Based on our modeling we speculate the reduction of functionality of the protein degradation mechanism as a possible competitive inhibition of the proteasome.     

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

Identification of Cytoplasmic and Nuclear Low-Molecular-Weight Heat-Shock Proteins in Tomato Fruit

Several low-molecular-weight heat-shock proteins were detectedby two-dimensional electro-phoresis in substantial amounts inboth nuclear and cytosolic extracts of tomato fruit. (Received September 16, 1992; Accepted November 27, 1992)     

Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations.     

2,4-Dichlorophenoxyacetic Acid-enhanced Phosphorylation of Soybean Nuclear Proteins

In vitro nuclear protein phosphorylation is enhanced in nuclei isolated from 2,4-dichlorophenoxyacetic acid (2,4-d)-treated mature soybean (Glycine max) hypocotyl relative to nuclei from untreated tissue. Increased nuclear protein phosphorylation correlates with increased levels of nuclear protein kinase activity. These changes generally parallel previously reported 2,4-d-enhanced RNA polymerase activity of these nuclei and the in vivo levels of RNA synthesis. Phosphate incorporation represents bona fide protein phosphorylation, with 87% of the label being identified as phosphoserine and 7% as phosphothreonine. Label from [γ-³²P]adenosine 5′-triphosphate is incorporated primarily into various nonhistone fractions with the greatest accumulation in loosely associated fractions (either released during incubation with ATP or removed by 0.15 m Nacl). Although electrophoretic analysis on sodium dodecyl sulfate gels shows no differences in the protein profiles of the loosely associated or sodium dodecyl sulfate-soluble nonhistone proteins, there are changes in the pattern of phosphorylation of other proteins, after 2,4-d treatment. Acid-soluble basic nuclear proteins are phosphorylated to a much lower extent than are the other nuclear protein fractions. While histone F₁ is subject to slight phosphorylation when nuclei are labeled in vitro, phosphorylation of the other histones is undetectable. One acid-soluble protein shows a substantial increase in quantity and in phosphorylation after 2,4-d treatment. This protein is similar in electrophoretic mobility to pea histone F₁ but its identity is unknown. Urea-acetic acid gels of the acid-soluble nuclear proteins show that auxin treatment results in increased quantities and in increased phosphorylation of various low mobility nonhistone basic nuclear proteins.     

Identification and Characterization of Mutations Affecting Sporulation in Saccharomyces Cerevisiae

Mutations affecting the synthesis of the sporulation amyloglucosidase were isolated in a homothallic strain of Saccharomyces cerevisiae, SCMS7-1. Two were found, both of which were deficient in sporulation at 34 degrees. One, SL484, sporulated to 50% normal levels at 30 degrees but less than 5% at 34 degrees or 22 degrees. The other, SL641, failed to sporulate at any temperature. Both mutants were blocked before premeiotic DNA synthesis, and both complemented spo1, spo3, and spo7. Genetic analysis of the mutation in SL484 indicated linkage to TRP5 and placed the gene 10 map units from TRP5 on chromosome VII. A plasmid containing an insert which complements the mutation in SL484 fails to complement SL641. We therefore conclude that these two mutations are in separate genes and we propose to call these genes SPO17 and SPO18. These two genes are (with SPO7, SPO8, and SPO9) among the earliest identified in the sporulation pathway and may interact directly with the positive and negative regulators RME and IME.     

A Cytoplasmic Suppressor of a Nuclear Mutation Affecting Mitochondrial Functions in Drosophila

Phenotypes relevant to oxidative phosphorylation (OXPHOS) in eukaryotes are jointly determined by nuclear and mitochondrial DNA (mtDNA). Thus, in humans, the variable clinical presentations of mitochondrial disease patients bearing the same primary mutation, whether in nuclear or mitochondrial DNA, have been attributed to putative genetic determinants carried in the “other” genome, though their identity and the molecular mechanism(s) by which they might act remain elusive. Here we demonstrate cytoplasmic suppression of the mitochondrial disease-like phenotype of the Drosophila melanogaster nuclear mutant tko^25t, which includes developmental delay, seizure sensitivity, and defective male courtship. The tko^25t strain carries a mutation in a mitoribosomal protein gene, causing OXPHOS deficiency due to defective intramitochondrial protein synthesis. Phenotypic suppression was associated with increased mtDNA copy number and increased mitochondrial biogenesis, as measured by the expression levels of porin voltage dependent anion channel and Spargel (PGC1α). Ubiquitous overexpression of Spargel in tko^25t flies phenocopied the suppressor, identifying it as a key mechanistic target thereof. Suppressor-strain mtDNAs differed from related nonsuppressor strain mtDNAs by several coding-region polymorphisms and by length and sequence variation in the noncoding region (NCR), in which the origin of mtDNA replication is located. Cytoplasm from four of five originally Wolbachia-infected strains showed the same suppressor effect, whereas that from neither of two uninfected strains did so, suggesting that the stress of chronic Wolbachia infection may provide evolutionary selection for improved mitochondrial fitness under metabolic stress. Our findings provide a paradigm for understanding the role of mtDNA genotype in human disease.     

Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Nuclear Basic Proteins of Xenopus laevis Sperm: Their Characterization and Synthesis during Spermatogenesis

Nuclear basic proteins from morphologically and functionally mature sperm of Xenopus laevis were analyzed by acid/urea/Triton X-100 polyacrylamide gel electrophoresis (AUT-PAGE). Six sperm-specific proteins (SP1-6) were identified in addition to somatic histones H3, H4 and smaller amount of H2A and H2B, but not H1. Of these, SP3–6 were unique in containing 33–41% arginine and having very low lysine/arginine ratios, while SP2 was more similar to H3 and H4 in having a lower arginine and higher lysine content. Fractionations of testicular cells at different spermatogenic stages by unit gravity sedimentation showed that primary spermatocytes and acrosomal vesicle spermatids possess typical somatic type histones but no SPs. Injection of [¹⁴C]-arginine into the testis and its tracing by fluorography on AUT-PAGE gels indicated that all somatic histones are synthesized during the stages between spermatogonia and primary spermatocytes, whereas SPs are synthesized at differentially regulated rates during the stages after acrosomal vesicle formation. In indirect immunofluorescence studies with anti-SP3-5 rabbit antiserum, a positive reaction was observed in the last step of spermiogenesis after the commencement of nuclear coiling.     

Relationship between Photosynthesis and Chlorophyll Content during Leaf Senescence of Rice Seedlings

Photosynthetic oxygen evolution, chlorophyll contents and chlorophylla /b ratios of 3rd to 6th leaves of rice seedlings were measuredto examine whether or not inactivation of photosynthesis duringsenescence is related to loss of chlorophyll. Photosyntheticactivity decreased more rapidly than chlorophyll content duringleaf senescence; as a result, the lower the leaf position, thelower was the rate of oxygen evolution determined on the basisof chlorophyll. Chlorophyll ab ratio also decreased with advancingsenescence. Electrophoretic analysis revealed that the declinein chlorophyll ab ratio is due to more rapid degradation ofthe reaction center complexes than light-harvesting chlorophyllab proteins of photosystem II and that the photosystem I reactioncenter disappears in parallel with the inactivation of photosynthesis.A simple method was developed to estimate the amounts of chlorophylla associated with the reaction center complexes of the two photosystemsfrom the total chlorophyll contents and chlorophyll ab ratiosof leaves. Rates of oxygen evolution, determined on the basisof chlorophyll a bound to the reaction center complexes, remainedconstant throughout the course of senescence. Thus, inactivationof photosynthesis is closely related with loss of the reactioncenter complexes during leaf senescence of rice seedlings. Theresults suggest that loss of photosynthesis is mainly causedby loss of a functional unit of photosynthesis or by a decreasein the number of whole chloroplasts. (Received April 22, 1987; Accepted August 13, 1987)     

Recovery and Characterization of Temperature-Sensitive Mutations Affecting Adult Viability in DROSOPHILA MELANOGASTER

Temperature-sensitive (ts) autonomous cell-lethal mutations have been used extensively to study important developmental phenomena, such as pattern formation, in Drosophila. Their utility would be enhanced considerably if it were possible to establish which cell type is primarily affected by each lesion. To facilitate such an approach we have isolated and characterized 21 EMS- induced X-linked adult-lethal (adl) mutants, 16 of which are ts. Most of these lesions also elicit ts lethal effects during preimaginal development. They represent 19 different loci distributed randomly along the X chromosome. The general properties of these mutations are described. In addition, results of an in-depth analysis (focus mapping and, in some cases, temperature shift and heat-pulse studies) of four strains, adl-1^ts1, sesE, adl-2^{ts 1} and rex are reported. Two major temperature-sensitive periods (TSPs) of adl-1 lethality were resolved: one during the second half of embryogenesis and the other coinciding with pupariation. Mosaic analysis revealed separate mesodermal foci for leg paralysis. Developmental analysis of adl-1 embryos suggest that the adl-1 product may be required for maintenance of muscle tissue. Two discrete TSPs of sesE lethality exist: one during the second instar and the other extending from late third instar to early pupation. Mosaic analysis of sesE lethality resolved a pair of neural foci, each of which appears to incorporate three separate foci for leg paralysis. Mosaic analysis of adl-2^{ts 1} revealed the existence of paired lethal foci that appear to map to the vicinity of the subesophageal ganglion. Analysis of rex mosaics resolved separate mesodermal foci for leg paralysis.     

Water Soluble Chlorophyll-Binding Proteins of Plants: Structure,Properties and Functions

Molecular Biology - Water soluble chlorophyll-binding proteins (WSCPs) of higher plants differ from most proteins containing chlorophyll or bacteriochlorophyll in that they are soluble in watr and...     

Bacteroids Are Stable during Dark-Induced Senescence of Soybean Root Nodules

Physiological and biochemical markers of metabolic competence were assayed in bacteroids isolated from root nodules of control, dark-stressed, and recovered plants of Glycine max Merr. cv `Woodworth.' Nitrogenase-dependent acetylene reduction by the whole plant decreased to 8% of control rates after 4 days of dark stress and could not be detected in plants dark stressed for 8 days. However, in bacteroids isolated anaerobically, almost 50% of initial acetylene reduction activity remained after 4 days of dark stress but was totally lost after 8 days of dark stress. Bacteroid acetylene reduction activity recovered faster than whole plant acetylene reduction activity when plants were dark stressed for 8 days and returned to a normal light regimen. Significant changes were not measured in bacteroid respiration, protein content, sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles, or in bacteroid proteolytic activity throughout the experiment. Immunoblots of bacteroid extracts revealed the presence of nitrogenase component II in control, 4-day dark-stressed, and 8-day dark-stressed plants that were allowed to recover under a normal light regimen, but not in 8-day dark-stressed plants. Our data indicate that dark stress does not greatly affect bacteroid metabolism or induce bacteroid senescence.     

Characterization of X-Linked Recessive Lethal Mutations Affecting Embryonic Morphogenesis in Drosophila Melanogaster

This study attempted to assay the zygotic contribution of X chromosome genes to the genetic control of embryonic morphogenesis in Drosophila melanogaster. A systematic screen for X-linked genes which affect the morphology of the embryo was undertaken, employing the phenotype of whole mount embryos as the major screening criterion. Of 800 EMS-induced lethal mutations analyzed, only 14% were embryonic lethal, and of these only a minority affected embryonic morphogenesis. By recombination and complementation analyses, the mutations that affected embryonic morphogenesis were sequestered into 26 complementation groups. Fourteen of the loci correspond to genes previously identified in a large-scale screen in which fixed cuticles were examined, and 12 new loci have been identified. Most of the mutations which disrupt embryonic morphology had specific and uniform mutant phenotypes. Mutations were recovered which disrupt major morphogenetic events such as gastrulation, germ band retraction and head involution. No mutations were found which arrest the embryos prior to blastoderm formation. However, a novel class was found, one comprised of mutations which interfere with the development of internal structures but not cuticular structures. Nevertheless, saturation of the X chromosome for genes important for embryonic morphogenesis is probably incomplete.     

Isolation and Characterization of Mutations Affecting the Transport of Hexose Phosphates in Escherichia coli

Mutants of Escherichia coli defective in the hexose phosphate transport system were isolated. Negative selection by penicillin treatment or positive selection with phosphonomycin was employed. These mutants grew normally on all carbon sources other than hexose phosphates. The map location of the mutations in 18 independently isolated mutant strains was investigated by transduction crosses. All of the mutations were found to lie in the same region of the chromosome, in the region represented by min 72 on the Taylor map. The order of the genes in this region was found to be mtl-cysE-pyrE-uhp-bgl-ilv. Revertants of some of the mutants exhibited altered regulatory control of this transport system.     

Photosynthesis and Other Traits in Relation to Chloroplast Number during Soybean Leaf Senescence

Soon after attaining full expansion, soybean (Glycine max [L.] Merr.) leaves enter a senescence phase marked by decline in photosynthetic rate and the progressive loss of chloroplast activity and composition. Our primary goal was to determine if this loss could be accounted for by sequential degradation of whole chloroplasts or by simultaneous degeneration of all chloroplasts. Total photosynthesis (TPs) measured as ¹⁴CO₂ uptake, chloroplast number, ribulose 1,5-bisphosphate carboxylase activity, uncoupled photosynthetic electron transport activity, soluble protein content, and chlorophyll content declined progressively during the 37 days after full leaf expansion. During this period, chloroplast number per unit leaf area was constant for all genotypes studied. We conclude that leaf senescence may be a two-stage process wherein the first stage chloroplast activity and composition declines, but chloroplast numbers do not change. During a brief terminal stage (11 days in our experiment), whole chloroplasts may be lost as well. As a second objective we wished to determine if variation in single-leaf total photosynthetic rate among soybean cultivars is related to corresponding variation in chloroplast number and/or chloroplast activity/composition. By comparing the means for three cultivars known to have rapid leaf TPs and for the three known to have slow TPs, we found the former group to be superior to the latter for all the previously mentioned leaf physiological traits. This superiority was related primarily to differences in chloroplast number and only secondarily to differences in activity and composition per chloroplast.     

Isolation and Characterization of the Multiple 7S Globulins of Soybean Proteins

Two major proteins (the 7S and 11S globulins) of soybean (Glycine max) were simultaneously isolated by a simple method based on their different solubilities in dilute tris (hydroxymethyl) aminomethane buffers. The purified 7S globulins, which represented essentially the entire 7S soybean protein fraction capable of dimerization at 0.1 ionic strength, were fractionated into five components by diethylaminoethyl Sephadex A-50 column chromatography. The five 7S components were characterized by disc-electrophoresis.