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Artykuły w czasopismach na temat "Cold Sensitive Phenotypes"
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Baliga, Chetana, Sandipan Majhi, Kajari Mondal, Antara Bhattacharjee, K. VijayRaghavan i Raghavan Varadarajan. "Rational elicitation of cold-sensitive phenotypes". Proceedings of the National Academy of Sciences 113, nr 18 (18.04.2016): E2506—E2515. http://dx.doi.org/10.1073/pnas.1604190113.
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Cold-sensitive phenotypes have helped us understand macromolecular assembly and biological phenomena, yet few attempts have been made to understand the basis of cold sensitivity or to elicit it by design. We report a method for rational design of cold-sensitive phenotypes. The method involves generation of partial loss-of-function mutants, at either buried or functional sites, coupled with selective overexpression strategies. The only essential input is amino acid sequence, although available structural information can be used as well. The method has been used to elicit cold-sensitive mutants of a variety of proteins, both monomeric and dimeric, and in multiple organisms, namelyEscherichia coli,Saccharomyces cerevisiae, andDrosophila melanogaster. This simple, yet effective technique of inducing cold sensitivity eliminates the need for complex mutations and provides a plausible molecular mechanism for eliciting cold-sensitive phenotypes.
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Novick, P., B. C. Osmond i D. Botstein. "Suppressors of yeast actin mutations." Genetics 121, nr 4 (1.04.1989): 659–74. http://dx.doi.org/10.1093/genetics/121.4.659.
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Abstract Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1+ genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17 degrees and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast.
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Flower, Ann M. "SecG Function and Phospholipid Metabolism inEscherichia coli". Journal of Bacteriology 183, nr 6 (15.03.2001): 2006–12. http://dx.doi.org/10.1128/jb.183.6.2006-2012.2001.
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ABSTRACT SecG is an auxiliary protein in the Sec-dependent protein export pathway of Escherichia coli. Although the precise function of SecG is unknown, it stimulates translocation activity and has been postulated to enhance the membrane insertion-deinsertion cycle of SecA. Deletion of secG was initially reported to result in a severe export defect and cold sensitivity. Later results demonstrated that both of these phenotypes were strain dependent, and it was proposed that an additional mutation was required for manifestation of the cold-sensitive phenotype. The results presented here demonstrate that the cold-sensitive secG deletion strain also contains a mutation in glpR that causes constitutive expression of the glp regulon. Introduction of both the glpRmutation and the secG deletion into a wild-type strain background produced a cold-sensitive phenotype, confirming the hypothesis that a second mutation (glpR) contributes to the cold-sensitive phenotype of secG deletion strains. It was speculated that the glpR mutation causes an intracellular depletion of glycerol-3-phosphate due to constitutive synthesis of GlpD and subsequent channeling of glycerol-3-phosphate into metabolic pathways. In support of this hypothesis, it was demonstrated that addition of glycerol-3-phosphate to the growth medium ameliorated the cold sensitivity, as did introduction of a glpD mutation. This depletion of glycerol-3-phosphate is predicted to limit phospholipid biosynthesis, causing an imbalance in the levels of membrane phospholipids. It is hypothesized that this state of phospholipid imbalance imparts a dependence on SecG for proper function or stabilization of the translocation apparatus.
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Nonet, M. L., i R. A. Young. "Intragenic and extragenic suppressors of mutations in the heptapeptide repeat domain of Saccharomyces cerevisiae RNA polymerase II." Genetics 123, nr 4 (1.12.1989): 715–24. http://dx.doi.org/10.1093/genetics/123.4.715.
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Abstract The largest subunit of RNA polymerase II contains a repeated heptapeptide sequence at its carboxy terminus. Yeast mutants with certain partial deletions of the carboxy-terminal repeat (CTR) domain are temperature-sensitive, cold-sensitive and are inositol auxotrophs. Intragenic and extragenic suppressors of the cold-sensitive phenotype of CTR domain deletion mutants were isolated and studied to investigate the function of this domain. Two types of intragenic suppressing mutations suppress the temperature-sensitivity, cold-sensitivity and inositol auxotrophy of CTR domain deletion mutants. Most intragenic mutations enlarge the repeat domain by duplicating various portions of the repeat coding sequence. Other intragenic suppressing mutations are point mutations in a conserved segment of the large subunit. An extragenic suppressing mutation (SRB2-1) was isolated that strongly suppresses the conditional and auxotrophic phenotypes of CTR domain mutations. The SRB2 gene was isolated and mapped, and an SRB2 partial deletion mutation (srb2 delta 10) was constructed. The srb2 delta 10 mutants are temperature-sensitive, cold-sensitive and are inositol auxotrophs. These phenotypes are characteristic of mutations in genes encoding components of the transcription apparatus. We propose that the SRB2 gene encodes a factor that is involved in RNA synthesis and may interact with the CTR domain of the large subunit of RNA polymerase II.
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Skiadopoulos, Mario H., Sonja Surman, Joanne M. Tatem, Maribel Paschalis, Shin-Lu Wu, Stephen A. Udem, Anna P. Durbin, Peter L. Collins i Brian R. Murphy. "Identification of Mutations Contributing to the Temperature-Sensitive, Cold-Adapted, and Attenuation Phenotypes of the Live-Attenuated Cold-Passage 45 (cp45) Human Parainfluenza Virus 3 Candidate Vaccine". Journal of Virology 73, nr 2 (1.02.1999): 1374–81. http://dx.doi.org/10.1128/jvi.73.2.1374-1381.1999.
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ABSTRACT The live-attenuated human parainfluenza virus 3 (PIV3) cold-passage 45 (cp45) candidate vaccine was shown previously to be safe, immunogenic, and phenotypically stable in seronegative human infants. Previous findings indicated that each of the three amino acid substitutions in the L polymerase protein of cp45 independently confers the temperature-sensitive (ts) and attenuation (att) phenotypes but not the cold-adaptation (ca) phenotype (29).cp45 contains 12 additional potentially important point mutations in other proteins (N, C, M, F, and hemagglutinin-neuraminidase [HN]) or in cis-acting sequences (the leader region and the transcription gene start [GS] signal of the N gene), and their contribution to these phenotypes was undefined. To further characterize the genetic basis for thets, ca, and att phenotypes of this promising vaccine candidate, we constructed, using a reverse genetics system, a recombinant cp45 virus that contained all 15 cp45-specific mutations mentioned above, and found that it was essentially indistinguishable from the biologically derived cp45 on the basis of plaque size, level of temperature sensitivity, cold adaptation, level of replication in the upper and lower respiratory tract of hamsters, and ability to protect hamsters from subsequent wild-type PIV3 challenge. We then constructed recombinant viruses containing the cp45 mutations in individual proteins as well as several combinations of mutations. Analysis of these recombinant viruses revealed that multiple cp45 mutations distributed throughout the genome contribute to the ts, ca, andatt phenotypes. In addition to the mutations in the L gene, at least one other mutation in the 3′ N region (i.e., including the leader, N GS, and N coding changes) contributes to thets phenotype. A recombinant virus containing all thecp45 mutations except those in L was more tsthan cp45, illustrating the complex nature of this phenotype. The ca phenotype of cp45 also is a complex composite phenotype, reflecting contributions of at least three separate genetic elements, namely, mutations within the 3′ N region, the L protein, and the C-M-F-HN region. The att phenotype is a composite of both ts and non-ts mutations. Attenuating ts mutations are located in the L protein, and non-ts attenuating mutations are located in the C and F proteins. The presence of multiple ts and non-ts attenuating mutations in cp45 likely contributes to the high level of attenuation and phenotypic stability of this promising vaccine candidate.
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Kuchka, Michael R., i Jonathan W. Jarvik. "Short-Flagella Mutants of Chlamydomonas reinhardtii". Genetics 115, nr 4 (1.04.1987): 685–91. http://dx.doi.org/10.1093/genetics/115.4.685.
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ABSTRACT Six short-flagella mutants were isolated by screening clones of mutagenized Chlamydomonas for slow swimmers. The six mutants identify three unlinked Mendelian genes, with three mutations in gene shf-1, two in shf-2 and one in shf-3. shf-1 and shf-2 have been mapped to chromosomes VI and I, respectively. Two of the shf-1 mutations have temperature-sensitive flagellar-assembly phenotypes, and one shf-2 mutant has a cold-sensitive phenotype. shf shf double mutants were constructed; depending on the alleles present they showed either flagellaless or short-flagella phenotypes. Phenotypic revertants of shf-1 and shf-2 mutants were isolated, and certain of them were found to carry extragenic suppressors, some dominant and some recessive. We suspect that the shf mutations affect components of a specific flagellar size-control system, the existence of which has been suggested by a variety of physiological experiments.
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Puziss, J. W., T. A. Hardy, R. B. Johnson, P. J. Roach i P. Hieter. "MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3". Molecular and Cellular Biology 14, nr 1 (styczeń 1994): 831–39. http://dx.doi.org/10.1128/mcb.14.1.831-839.1994.
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The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.
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Puziss, J. W., T. A. Hardy, R. B. Johnson, P. J. Roach i P. Hieter. "MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3." Molecular and Cellular Biology 14, nr 1 (styczeń 1994): 831–39. http://dx.doi.org/10.1128/mcb.14.1.831.
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The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3.
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Fane, B. A., i M. Hayashi. "Second-site suppressors of a cold-sensitive prohead accessory protein of bacteriophage phi X174." Genetics 128, nr 4 (1.08.1991): 663–71. http://dx.doi.org/10.1093/genetics/128.4.663.
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Abstract This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.
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Hecht, Ralph M., Mary A. Norman, Tammy Vu i William Jones. "A novel set of uncoordinated mutants inCaenorhabditis elegansuncovered by cold-sensitive mutations". Genome 39, nr 2 (1.04.1996): 459–64. http://dx.doi.org/10.1139/g96-058.
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A set of uncoordinated (Unc) cold-sensitive (cs) mutants was isolated at a stringent condition of 11 °C. About half of the 13 independently isolated cs-Unc mutants were alleles of three X-linked Unc mutants that exhibited the "kinker" phenotype. The remaining four isolates identified new mutants that exhibited "kinker," "coiler," or severe paralytic phenotypes. The temperature-sensitive period (TSP) for each gene was determined. As a homozygous or heterozygous dominant, unc-125 exhibited a TSP throughout all stages of development. Its severe paralysis was immediately observed upon a shift down to 11 °C and reversed upon a shift up to 23 °C. The reversible thermolability of the unc-125 gene product indicated that it may function in a multicomponent process involved in neuro-excitation. Key words : Caenorhabditis elegans, cold-sensitive uncoordinated mutants, cs-Unc.
Rozprawy doktorskie na temat "Cold Sensitive Phenotypes"
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Chetana, Baliga B. "Rational Elicitation of Cold Sensitive Phenotypes". Thesis, 2015. http://etd.iisc.ac.in/handle/2005/4081.
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Conditional gene expression and conditional mutants provide a means to modulate the expression of specific genes and to control the activity of their protein products in vivo, so as to be able to study their effects on the cell. Conditional mutants are functional under one set of conditions, termed ‘permissive’, while under other conditions that are ‘restrictive’, they become non-functional. The wild-type (Wt) is functional under both these conditions. Conditional mutants are especially useful for studying essential or lethal genes in an organism. In the case of temperature-sensitive (ts) and cold-sensitive mutants (cs), by using temperature shift as a condition, the target gene function can be modulated easily, rapidly, reversibly and selectively, at any stage in the life cycle of an organism. Cs mutants are less common and molecular determinants of cs phenotypes are poorly understood.This thesis presents a method for rational elicitation of cold sensitive phenotypes which involves design of partial loss-of-function mutants based solely on amino acid sequence, and coupling of such mutants to a heat responsive promoter to result in cs phenotypes. This approach has been validated for different proteins (CcdB, Gal4, Ura3 and Trp1) in different organisms (E. coli, S. cerevisiae and D. melanogaster). This is a straightforward approach which does not involve complex temperature-dependent, mutational effects.Additional characterization of purified Gal4 mutants by measuring protein thermal stability and DNA binding affinity, as well as measurements of transcript levels by qPCR were carried out, to understand the molecular basis of the cs phenotype.
The pBAD series of vectors, containing the PBAD promoter and the araC regulator-activator, are very convenient for cloning and expression purposes. They have been widely for cloning and for graded expression of cloned genes. However, there have been reports of non-uniform gene expression across cells at sub-saturating concentrations of arabinose. This thesis also covers studies on this issue of heterogeneity of expression from PBAD promoters at the single cell level, using stable and degradable GFPs as reporters, in a variety of conditions such as constitutive versus autocatalytic expression of arabinose transporter, presence and absence of arabinose metabolising araBAD genes in the host, and varying time periods of induction.
Several single amino acid substitutions which cause folding defects in the protein are seen to give rise to cs or ts phenotypes. The key to understanding the basis of such phenotypes caused by folding defective mutants lies in the folding pathway of the protein. To this end, the folding kinetics of E. coli CcdB were studied, which is prerequisite for understanding the folding defects in various ts/cs mutants of CcdB. This study looks at the folding of a dimeric protein, which is of great interest as it involves conformational changes as well as association steps. The results from various experimental observations, ligand binding studies and simulations lead to the conclusion that CcdB folds via parallel pathways, each involving an unstructured dimeric intermediate, to arrive at its native state.
In summary, the thesis covers the research carried out ontuning conditional gene expression, rationally designing conditional mutants and understanding their folding mechanisms.
Części książek na temat "Cold Sensitive Phenotypes"
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West-Eberhard, Mary Jane. "Maintenance without Equilibrium". W Developmental Plasticity and Evolution. Oxford University Press, 2003. http://dx.doi.org/10.1093/oso/9780195122343.003.0029.
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The old question of the maintenance of alternative phenotypes appears in a new light when seen from a developmental point of view. In the past, alternatives were treated as genetic polymorphisms and explained in terms of genetic equilibria. During the synthesis of genetics and evolutionary theory in the early twentieth century, single-locus and linkagegroup genetic polymorphisms were seen as maintained in three different ways: by heterosis (heterozygote advantage), by frequency-dependent selection, and by balancing selection in a spatially heterogeneous environment (summarized in Dobzhansky, 1970). Later models showed how alternative behavioral and morphological tactics in theory could be maintained as “evolutionary stable strategies” (ESSs; see summary and history in Parker, 1984a). ESS models treat the maintenance of alternatives not as a problem in genetics but as a problem in game theory. This chapter views the maintenance of alternative phenotypes as a problem in the evolution of regulation, where the genes responsible for the maintenance of alternatives influence regulation at condition-sensitive, polygenic developmental decision points whose thresholds can be adjusted upward and downward under selection. These adjustments affect the ratio of alternatives observed. Regulation may sometimes be determined by a small number of alleles, but even then, insofar as most readily observed alternative phenotypes are concerned, they are alleles that influence a polygenic switch (see chapter 6). Rephrased in developmental terms, the question of maintenance of alternative phenotypes, whether genotype specific or conditional, becomes: What selective factors can influence the evolution of regulatory mechanisms so as to preserve the expression of two or more alternative traits rather than just one? Along with the rest of this book, this chapter focuses on the importance of developmental plasticity for evolutionary interpretations. It describes three types of regulation that have been assumed by theoretical models, and argues that two of them—genotype-specific and stochastic regulation— are relatively uncommon in nature, even though common in theoretical research. I then discuss reasons for the superior stability of the third, conditional type of regulation and argue that the properties of complex adaptive alternative phenotypes may help explain the maintenance of genetic polymorphisms.
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"The principle of the MAIEA technique depends on the binding of two antibodies made in different species to different determinants on the same membrane component to form of a tri-molecular complex [4]. Briefly, a murine monoclonal antibody (MAb) and human antibody are incubated simultaneously with red cells. Excess antibody is removed, the sensitized cells are solubilised with Triton, so the tri-molecular complex is released into solution. The complex is detected by an ELISA type assay. The tri-molecular complex is captured by an anti-mouse globulin precoated onto a microtitre plate. The human antibody is then detected by a peroxidase-conjugated anti-human IgG. A positive reaction gives a high absorbance value and a negative reaction gives a low absorbance value. A negative result is obtained when the antibodies used bind to different membrane components, so no tri-molecular complex is formed. A negative result is also obtained when the monoclonal antibody and human antibody compete for the same epitope. Results can be represented as ratios of absorbances for antigen positive to antigen negative cells or as bar charts. In these studies a murine anti-CR1 (E11) and human anti-Kna and other Knops system antibodies were used against antigen positive and antigen negative cells. Absorbances for antigen positive cells with anti-Kna, anti-McCa anti-Sla and anti-Yka were high and results for the antigen-negative cells were low [8]. Comparison of chymotrypsin treated Kn(a+) cells with Kn(a-) cells showed that chymotrypsin did indeed destroy Kna antigen; chymotrypsin treated cells, therefore, were suitable cells to use as antigen negative cells when cells of rare phenotype were not available [8]. These reactions gave significantly positive ratios (Table I). In contrast, low absorbances were recorded for Cs(a+) and Cs(a-) cells with anti-Csa, the 1:1 ratio indicating a negative result (Table I). Serologically the Helgeson phenotype cells have a Knops null phenotype, all 4 antigens are negative but the antigens could be detected by flow cytometry and in immune precipitation [6,7]. Moulds and colleagues provided an explanation for this when they found that such cells did not completely lack CR1 but had a low copy number of CR1 molecules per cell [9]. Had it not been known already, the presence of Knops system antigens on Helgeson phenotype cells could have been deduced from the MAIEA results. The absorbance values for Helgeson phenotype cells were significantly higher than for antigen negative cells for Kna, McCa and Yka [8]. MAIEA has confirmed that Kna, McCa, Sla and Yka but not Csa are associated with the CR1 molecule in the red cell membrane and can detect weak expression of CR1 antigens on Helgeson phenotype cells [8]. MAIEA is useful for investigating problem antibodies suspected to be Knops system antibodies and can also be used to Knops phenotype cells with poor expression of Knops system antigens." W Transfusion Immunology and Medicine, 189. CRC Press, 1995. http://dx.doi.org/10.1201/9781482273441-8.
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Neitz, Jay, Maureen Neitz i Gerald H. Jacobs. "More than three cone types in normal color vision?" W OSA Annual Meeting. Washington, D.C.: Optica Publishing Group, 1990. http://dx.doi.org/10.1364/oam.1990.fm6.
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Normal human color vision is usually thought to be based on only three spectrally different cone types. However, two facts suggest the possibility that some color-normal males could have more than three cone pigment types: (1) Most people with normal color vision have more than two photopigment genes on each X-chromosome and (2) there appear to be genetically specified variations in spectral positions of the normal middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) pigments. For example, a male might have one gene encoding an LWS pigment and two genes encoding slightly different MWS pigments. If all three different X-encoded genes were expressed in different cones, then this person would have four spectrally different cone types. How firm is the assumption that more than two of the X-encoded pigment genes can be expressed? Both analysis of the statistics of photopigment gene number among different color vision phenotypes and analysis of the arrangement of pigment genes on the X-chromosome yield insight into this aspect of photopigment gene expression. These analyses suggest that individuals with multiple pigment genes on the X-chromosome may express more than two of those genes.
Raporty organizacyjne na temat "Cold Sensitive Phenotypes"
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Crisosto, Carlos, Susan Lurie, Haya Friedman, Ebenezer Ogundiwin, Cameron Peace i George Manganaris. Biological Systems Approach to Developing Mealiness-free Peach and Nectarine Fruit. United States Department of Agriculture, 2007. http://dx.doi.org/10.32747/2007.7592650.bard.
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Peach and nectarine production worldwide is increasing; however consumption is flat or declining because of the inconsistent eating quality experienced by consumers. The main factor for this inconsistent quality is mealiness or woolliness, a form of chilling injury that develops following shipping periods in the global fruit market today. Our research groups have devised various postharvest methods to prolong storage life, including controlled atmosphere and delayed storage; however, these treatments only delay mealiness. Mealiness texture results from disruption of the normal ripening process involving disassembly of cell wall material, and creates a soft fruit texture that is dry and grainy instead of juicy and smooth. Solving this problem is a prerequisite for increasing the demand for fresh peach and nectarine. Two approaches were used to reveal genes and their associated biochemical processes that can confer resistance to mealiness or wooliness. At the Volcani Center, Israel, a nectarine cultivar and the peach cultivar (isogenetic materials) from which the nectarine cultivar spontaneously arose, and at the Kearney Agricultural Center of UC Davis, USA, a peach population that segregates for quantitative resistance to mealiness was used for dissecting the genetic components of mealiness development. During our project we have conducted research integrating the information from phenotypic, biochemical and gene expression studies, proposed possible candidate genes and SNPs-QTLs mapping that are involved in reducing peach mealiness susceptibility. Numerous genes related to ethylene biosynthesis and its signal transduction, cell wall structure and metabolism, stress response, different transcription factor families were detected as being differentially accumulated in the cold-treated samples of these sensitive and less sensitive genotypes. The ability to produce ethylene and keep active genes involved in ethylene signaling, GTP-binding protein, EIN-3 binding protein and an ethylene receptor and activation of ethyleneresponsive fruit ripening genes during cold storage provided greater resistance to CI. Interestingly, in the functional category of genes differentially expressed at harvest, less chilling sensitive cultivar had more genes in categories related to antioxidant and heat sock proteins/chaperones that may help fruit to adapt to low temperature stress. The specific objectives of the proposed research were to: characterize the phenotypes and cell wall components of the two resistant systems in response to mealiness- inducing conditions; identify commonalities and specific differences in cell wall proteins and the transcriptome that are associated with low mealiness incidence; integrate the information from phenotypic, biochemical, and gene expression studies to identify candidate genes that are involved in reducing mealiness susceptibility; locate these genes in the Prunus genome; and associate the genes with genomic regions conferring quantitative genetic variation for mealiness resistance. By doing this we will locate genetic markers for mealiness development, essential tools for selection of mealiness resistant peach lines with improved fruit storability and quality. In our research, QTLs have been located in our peach SNPs map, and proposed candidate genes obtained from the integrated result of phenotypic, biochemical and gene expression analysis are being identified in our QTLs as an approach searching for consistent assistant markers for peach breeding programs.
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Lers, Amnon, Majid R. Foolad i Haya Friedman. genetic basis for postharvest chilling tolerance in tomato fruit. United States Department of Agriculture, styczeń 2014. http://dx.doi.org/10.32747/2014.7600014.bard.
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ABSTRACT Postharvest losses of fresh produce are estimated globally to be around 30%. Reducing these losses is considered a major solution to ensure global food security. Storage at low temperatures is an efficient practice to prolong postharvest performance of crops with minimal negative impact on produce quality or human health and the environment. However, many fresh produce commodities are susceptible to chilling temperatures, and the application of cold storage is limited as it would cause physiological chilling injury (CI) leading to reduced produce quality. Further, the primary CI becomes a preferred site for pathogens leading to decay and massive produce losses. Thus, chilling sensitive crops should be stored at higher minimal temperatures, which curtails their marketing life and in some cases necessitates the use of other storage strategies. Development of new knowledge about the biological basis for chilling tolerance in fruits and vegetables should allow development of both new varieties more tolerant to cold, and more efficient postharvest storage treatments and storage conditions. In order to improve the agricultural performance of modern crop varieties, including tomato, there is great potential in introgression of marker-defined genomic regions from wild species onto the background of elite breeding lines. To exploit this potential for improving tomato fruit chilling tolerance during postharvest storage, we have used in this research a recombinant inbred line (RIL) population derived from a cross between the red-fruited tomato wild species SolanumpimpinellifoliumL. accession LA2093 and an advanced Solanum lycopersicumL. tomato breeding line NCEBR-1, developed in the laboratory of the US co-PI. The original specific objectives were: 1) Screening of RIL population resulting from the cross NCEBR1 X LA2093 for fruit chilling response during postharvest storage and estimation of its heritability; 2) Perform a transcriptopmic and bioinformatics analysis for the two parental lines following exposure to chilling storage. During the course of the project, we learned that we could measure greater differences in chilling responses among specific RILs compared to that observed between the two parental lines, and thus we decided not to perform transcriptomic analysis and instead invest our efforts more on characterization of the RILs. Performing the transcriptomic analysis for several RILs, which significantly differ in their chilling tolerance/sensitivity, at a later stage could result with more significant insights. The RIL population, (172 lines), was used in field experiment in which fruits were examined for chilling sensitivity by determining CI severity. Following the field experiments, including 4 harvest days and CI measurements, two extreme tails of the response distribution, each consisting of 11 RILs exhibiting either high sensitivity or tolerance to chilling stress, were identified and were further examined for chilling response in greenhouse experiments. Across the RILs, we found significant (P < 0.01) correlation between field and greenhouse grown plants in fruit CI. Two groups of 5 RILs, whose fruits exhibited reproducible chilling tolerant/sensitive phenotypes in both field and greenhouse experiments, were selected for further analyses. Numerous genetic, physiological, biochemical and molecular variations were investigated in response to postharvest chilling stress in the selected RILs. We confirmed the differential response of the parental lines of the RIL population to chilling stress, and examined the extent of variation in the RIL population in response to chilling treatment. We determined parameters which would be useful for further characterization of chilling response in the RIL population. These included chlorophyll fluorescence Fv/Fm, water loss, total non-enzymatic potential of antioxidant activity, ascorbate and proline content, and expression of LeCBF1 gene, known to be associated with cold acclimation. These parameters could be used in continuation studies for the identification and genetic mapping of loci contributing to chilling tolerance in this population, and identifying genetic markers associated with chilling tolerance in tomato. Once genetic markers associated with chilling tolerance are identified, the trait could be transferred to different genetic background via marker-assisted selection (MAS) and breeding. The collaborative research established in this program has resulted in new information and insights in this area of research and the collaboration will be continued to obtain further insights into the genetic, molecular biology and physiology of postharvest chilling tolerance in tomato fruit. The US Co-PI, developed the RIL population that was used for screening and measurement of the relevant chilling stress responses and conducted statistical analyses of the data. Because we were not able to grow the RIL population under field conditions in two successive generations, we could not estimate heritability of response to chilling temperatures. However, we plan to continue the research, grow the RIL progeny in the field again, and determine heritability of chilling tolerance in a near future. The IS and US investigators interacted regularly and plan to continue and expand on this study, since combing the expertise of the Co-PI in genetics and breeding with that of the PI in postharvest physiology and molecular biology will have great impact on this line of research, given the significant findings of this one-year feasibility project.
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Horwitz, Benjamin, i Barbara Gillian Turgeon. Secondary Metabolites, Stress, and Signaling: Roles and Regulation of Peptides Produced by Non-ribosomal Peptide Synthetases. United States Department of Agriculture, 2005. http://dx.doi.org/10.32747/2005.7696522.bard.
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Fungal pathogens of plants produce a diverse array of small molecules. Often referred to as secondary metabolites because they were thought to be dispensable for basic functions, they may indeed have central roles as signals for the fungal cell, and in interactions with the host. We have identified more than a dozen genes encoding nonribosomal peptide synthetases (NPS) in Cochliobolusheterostrophus, the agent of southern corn leaf blight. The aim of this project was to identify roles of these genes in stress responses and signaling. The first objective was to test a complete collection of C. heterostrophus nonribosomal peptide synthetase (NRPS)-encoding gene deletion mutant and wildtype (WT) strains for sensitivity to various agents of oxidative (ROS) and nitrosative (RNOS) stress, in vitro. The second objective and next step in this part of the project was to study the relevance of sensitivity to ROS and RNOS in the host pathogen interaction, by measuring the production of ROS and RNOS in planta, when plants are inoculated with wild type and mutant strains. A third objective was to study expression of any genes shown to be involved in sensitivity to ROS or RNOS, in vitro and in planta. Another objective was to determine if any of the genes involved in oxidative or nitrosative stress responses are regulated by components of signal transduction pathways (STP) that we have identified and to determine where mechanisms overlap. Study of the collection of nps mutants identified phenotypes relevant for virulence, development and oxidative stress resistance for two of the genes, NPS2 and NPS6. Mutants in genes related to RNOS stress have no virulence phenotypes, while some of those related to ROS stress have reduced virulence as well as developmental phenotypes, so we focused primarily on ROS stress pathways. Furthermore, the identification of NPS2 and NPS6 as encoding for NRPS responsible for siderophore biosynthesis lent a new focus to the project, regulation by Fe. We have not yet developed good methods to image ROS in planta and work in this direction is continuing. We found that NPS6 expression is repressed by Fe, responding over the physiological Fe concentration range. Studying our collection of mutants, we found that conserved MAPK and G protein signal transduction pathways are dispensable for Fe regulation of NPS6, and initiated work to identify other pathways. The transcription factor SreA is one candidate, and is responsible for part, but not all, of the control of NPS6 expression. The results of this project show that the pathogen contends with oxidative stress through several signaling pathways. Loss of the siderophore produced by Nps6 makes the fungus sensitive to oxidative stress, and decreases virulence, suggesting a central role of the ability to sequester and take up extracellular iron in the host-pathogen interaction. Siderophores, and manipulation of Fe levels, could be targets for new strategies to deal with fungal pathogens of maize and other plants.
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Horwitz, Benjamin A., i Barbara Gillian Turgeon. Fungal Iron Acquisition, Oxidative Stress and Virulence in the Cochliobolus-maize Interaction. United States Department of Agriculture, marzec 2012. http://dx.doi.org/10.32747/2012.7709885.bard.
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Our project focused on genes for high affinity iron acquisition in Cochliobolus heterostrophus, a necrotrophic pathogen of maize, and their intertwined relationship to oxidative stress status and virulence of the fungus on the host. An intriguing question was why mutants lacking the nonribosomal peptide synthetase (NRPS) gene (NPS6) responsible for synthesis of the extracellular siderophore, coprogen, are sensitive to oxidative stress. Our overall objective was to understand the mechanistic connection between iron stress and oxidative stress as related to virulence of a plant pathogen to its host. The first objective was to examine the interface where small molecule peptide and reactive oxygen species (ROS) mechanisms overlap. The second objective was to determine if the molecular explanation for common function is common signal transduction pathways. These pathways, built around sensor kinases, response regulators, and transcription factors may link sequestering of iron, production of antioxidants, resistance to oxidative stress, and virulence. We tested these hypotheses by genetic manipulation of the pathogen, virulence assays on the host plant, and by following the expression of key fungal genes. An addition to the original program, made in the first year, was to develop, for fungi, a genetically encoded indicator of redox state based on the commercially available Gfp-based probe pHyper, designed for animal cell biology. We implemented several tools including a genetically encoded indicator of redox state, a procedure to grow iron-depleted plants, and constructed a number of new mutants in regulatory genes. Lack of the major Fe acquisition pathways results in an almost completely avirulent phenotype, showing how critical Fe acquisition is for the pathogen to cause disease. Mutants in conserved signaling pathways have normal ability to regulate NPS6 in response to Fe levels, as do mutants in Lae1 and Vel1, two master regulators of gene expression. Vel1 mutants are sensitive to oxidative stress, and the reason may be underexpression of a catalase gene. In nps6 mutants, CAT3 is also underexpressed, perhaps explaining the sensitivity to oxidative stress. We constructed a deletion mutant for the Fe sensor-regulator SreA and found that it is required for down regulation of NPS6 under Fe-replete conditions. Lack of SreA, though, did not make the fungus over-sensitive to ROS, though the mutant had a slow growth rate. This suggests that overproduction of siderophore under Fe-replete conditions is not very damaging. On the other hand, increasing Fe levels protected nps6 mutants from inhibition by ROS, implying that Fe-catalyzed Fenton reactions are not the main factor in its sensitivity to ROS. We have made some progress in understanding why siderophore mutants are sensitive to oxidative stress, and in doing so, defined some novel regulatory relationships. Catalase genes, which are not directly related to siderophore biosynthesis, are underexpressed in nps6 mutants, suggesting that the siderophore product (with or without bound Fe) may act as a signal. Siderophores, therefore, could be a target for intervention in the field, either by supplying an incorrect signal or blocking a signal normally provided during infection. We already know that nps6 mutants cause smaller lesions and have difficulty establishing invasive growth in the host. Lae1 and Vel1 are the first factors shown to regulate both super virulence conferred by T-toxin, and basic pathogenicity, due to unknown factors. The mutants are also altered in oxidative stress responses, key to success in the infection court, asexual and sexual development, essential for fungal dissemination in the field, aerial hyphal growth, and pigment biosynthesis, essential for survival in the field. Mutants in genes encoding NADPH oxidase (Nox) are compromised in development and virulence. Indeed the triple mutant, which should lack all Nox activity, was nearly avirulent. Again, gene expression experiments provided us with initial evidence that superoxide produced by the fungus may be most important as a signal. Blocking oxidant production by the pathogen may be a way to protect the plant host, in interactions with necrotrophs such as C. heterostrophus which seem to thrive in an oxidant environment.
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Meir, Shimon, Michael S. Reid, Cai-Zhong Jiang, Amnon Lers i Sonia Philosoph-Hadas. Molecular Studies of Postharvest Leaf and Flower Senescence. United States Department of Agriculture, styczeń 2011. http://dx.doi.org/10.32747/2011.7592657.bard.
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Original objectives: To understand the regulation of abscission by exploring the nature of changes of auxin-related gene expression in tomato (Lycopersicon esculatumMill) abscission zones (AZs) following organ removal, and by analyzing the function of these genes. Our specific goals were: 1) To complete the microarray analyses in tomato flower and leaf AZs, for identifying genes whose expression changes early in response to auxin depletion; 2) To examine, using virus-induced gene silencing (VIGS), the effect of silencing target genes on ethylene sensitivity and abscission competence of the leaf and flower AZs; 3) To isolate and characterize promoters from AZ-specific genes to be used in functional analysis; 4) To generate stable transgenic tomato plants with selected genes silenced with RNAi, under the control of an AZ-specific promoter, for further characterization of their abscission phenotypes. Background: Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many ornamentals and other fresh produce. The process is initiated by changes in the auxin gradient across the AZ, and is triggered by ethylene. Although changes in gene expression have been correlated with the ethylene-mediated execution of abscission, there is almost no information on the initiation of the abscission process, as the AZ becomes sensitized to ethylene. The present project was focused on elucidating these early molecular regulatory events, in order to gain a better control of the abscission process for agricultural manipulations. Major conclusions, solutions, achievements: Microarray analyses, using the Affymetrix Tomato GeneChip®, revealed changes in expression, occurring early in abscission, of many genes with possible regulatory functions. These included a range of auxin- and ethylene-related transcription factors (TFs), other TFs that are transiently induced just after flower removal, and a set of novel AZ-specific genes. We also identified four different defense-related genes, including: Cysteine-type endopeptidase, α- DOX1, WIN2, and SDF2, that are newly-associated with the late stage of the abscission process. This supports the activation of different defense responses and strategies at the late abscission stages, which may enable efficient protection of the exposed tissue toward different environmental stresses. To facilitate functional studies we implemented an efficient VIGS system in tomato, and isolated two abscission-specific promoters (pTAPG1 and pTAPG4) for gene silencing in stable transformation. Using the VIGS system we could demonstrate the importance of TAPGs in abscission of tomato leaf petioles, and evaluated the importance of more than 45 genes in abscission. Among them we identified few critical genes involved in leaf and flower abscission. These included: PTRP-F1, PRP, TKN4, KNOTTED-like homeobox TF, KD1, and KNOX-like homeodomain protein genes, the silencing of which caused a striking retardation of pedicel abscission, and ERF1, ERF4, Clavata-like3 protein, Sucrose transporter protein, and IAA10 genes, the silencing of which delayed petiole abscission. The importance of PRPand KD1 genes in abscission was confirmed also by antisense–silencing using pTAPG4. Experiments testing the effects of RNAi silencing of few other genes are still in progress, The analysis of the microarray results of flower and leaf AZs allowed us to establish a clear sequence of events occurring during acquisition of tissue sensitivity to ethylene, and to confirm our hypothesis that acquisition of ethylene sensitivity in the AZ is associated with altered expression of auxin-regulated genes in both AZs. Implication, both scientific and agricultural: Our studies had provided new insights into the regulation of the abscission process, and shaded light on the molecular mechanisms that drive the acquisition of abscission competence in the AZ. We pointed out some critical genes involved in regulation of abscission, and further expanded our knowledge of auxin-ethylene cross talk during the abscission process. This permits the development of novel techniques for manipulating abscission, and thereby improving the postharvest performance of ornamentals and other crops.
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Granot, David, Richard Amasino i Avner Silber. Mutual effects of hexose phosphorylation enzymes and phosphorous on plant development. United States Department of Agriculture, styczeń 2006. http://dx.doi.org/10.32747/2006.7587223.bard.
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Research objectives 1) Analyze the combined effects of hexose phosphorylation and P level in tomato and Arabidopsis plants 2) Analyze the combined effects of hexose phosphorylation and P level in pho1 and pho2 Arabidopsis mutants 3) Clone and analyze the PHO2 gene 4) Select Arabidopsis mutants resistant to high and low P 5) Analyze the Arabidopsis mutants and clone the corresponding genes 6) Survey wild tomato species for growth characteristics at various P levels Background to the topic Hexose phosphorylating enzymes, the first enzymes of sugar metabolism, regulate key processes in plants such as photosynthesis, growth, senescence and vascular transport. We have previously discovered that hexose phosphorylating enzymes might regulate these processes as a function of phosphorous (P) concentration, and might accelerate acquisition of P, one of the most limiting nutrients in the soil. These discoveries have opened new avenues to gain fundamental knowledge about the relationship between P, sugar phosphorylation and plant development. Since both hexose phosphorylating enzymes and P levels affect plant development, their interaction is of major importance for agriculture. Due to the acceleration of senescence caused by the combined effects of hexose phosphorylation and P concentration, traits affecting P uptake may have been lost in the course of cultivation in which fertilization with relatively high P (30 mg/L) are commonly used. We therefore intended to survey wild tomato species for high P-acquisition at low P soil levels. Genetic resources with high P-acquisition will serve not only to generate a segregating population to map the trait and clone the gene, but will also provide a means to follow the trait in classical breeding programs. This approach could potentially be applicable for other crops as well. Major conclusions, solutions, achievements Our results confirm the mutual effect of hexose phosphorylating enzymes and P level on plant development. Two major aspects of this mutual effect arose. One is related to P toxicity in which HXK seems to play a major role, and the second is related to the effect of HXK on P concentration in the plant. Using tomato plants we demonstrated that high HXK activity increased leaf P concentration, and induced P toxicity when leaf P concentration increases above a certain high level. These results further support our prediction that the desired trait of high-P acquisition might have been lost in the course of cultivation and might exist in wild species. Indeed, in a survey of wild species we identified tomato species that acquired P and performed better at low P (in the irrigation water) compared to the cultivated Lycopersicon esculentum species. The connection between hexose phosphorylation and P toxicity has also been shown with the P sensitive species VerticordiaplumosaL . in which P toxicity is manifested by accelerated senescence (Silber et al., 2003). In a previous work we uncovered the phenomenon of sugar induced cell death (SICD) in yeast cells. Subsequently we showed that SICD is dependent on the rate of hexose phosphorylation as determined by Arabidopsis thaliana hexokinase. In this study we have shown that hexokinase dependent SICD has many characteristics of programmed cell death (PCD) (Granot et al., 2003). High hexokinase activity accelerates senescence (a PCD process) of tomato plants, which is further enhanced by high P. Hence, hexokinase mediated PCD might be a general phenomena. Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including tomato. Senescing leaves are particularly susceptible to B. cinerea infection and delaying leaf senescence might reduce this susceptibility. It has been suggested that B. cinerea’s mode of action may be based on induction of precocious senescence. Using tomato plants developed in the course of the preceding BARD grant (IS 2894-97) and characterized throughout this research (Swartzberg et al., 2006), we have shown that B. cinerea indeed induces senescence and is inhibited by autoregulated production of cytokinin (Swartzberg et al., submitted). To further determine how hexokinase mediates sugar effects we have analyzed tomato plants that express Arabidopsis HXK1 (AtHXK1) grown at different P levels in the irrigation water. We found that Arabidopsis hexokinase mediates sugar signalling in tomato plants independently of hexose phosphate (Kandel-Kfir et al., submitted). To study which hexokinase is involved in sugar sensing we searched and identified two additional HXK genes in tomato plants (Kandel-Kfir et al., 2006). Tomato plants have two different hexose phosphorylating enzymes; hexokinases (HXKs) that can phosphorylate either glucose or fructose, and fructokinases (FRKs) that specifically phosphorylate fructose. To complete the search for genes encoding hexose phosphorylating enzymes we identified a forth fructokinase gene (FRK) (German et al., 2004). The intracellular localization of the four tomato HXK and four FRK enzymes has been determined using GFP fusion analysis in tobacco protoplasts (Kandel-Kfir et al., 2006; Hilla-Weissler et al., 2006). One of the HXK isozymes and one of the FRK isozymes are located within plastids. The other three HXK isozymes are associated with the mitochondria while the other three FRK isozymes are dispersed in the cytosol. We concluded that HXK and FRK are spatially separated in plant cytoplasm and accordingly might play different metabolic and perhaps signalling roles. We have started to analyze the role of the various HXK and FRK genes in plant development. So far we found that LeFRK2 is required for xylem development (German et al., 2003). Irrigation with different P levels had no effect on the phenotype of LeFRK2 antisense plants. In the course of this research we developed a rapid method for the analysis of zygosity in transgenic plants (German et al., 2003).