Agricultural and Biological Sciences › Plant Science

Plant Genetic and Mutation Studies

Works Count: 31377

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This cluster of papers focuses on mutation breeding techniques such as TILLING and induced mutations to improve crops through functional genomics, genetic variation, and the use of ionizing radiation. The research explores methods for identifying point mutations, genome-wide sequencing, and the application of reverse genetics for plant improvement.

Keywords

Mutation Breeding; TILLING; Induced Mutations; Functional Genomics; Plant Improvement; Genetic Variation; Ionizing Radiation; Reverse Genetics; Point Mutations; Genome-wide Sequencing

Targeted screening for induced mutations

2000-04-01

Claire M. McCallum , Luca Comai , Elizabeth A. Greene +1 more

Mitosis and the Physiology of Cell Division

1961-01-01

Daniel Mazia

Microsatellite Libraries Enriched for Several Microsatellite Sequences in Plants

1996-05-01

Keith J. Edwards , J. H. A. Barker , Adrian M. Daly +2 more

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Vicia faba bioassay for environmental toxicity monitoring: A review

2015-09-27

Munawar Iqbal

Improvement of stress tolerance of wheat and barley by modulation of expression of DREB/CBF factors

2010-07-20

Sarah Morran , Omid Eini , Tatiana Pyvovarenko +7 more

Transcription factors have been shown to control the activity of multiple stress response genes in a coordinated manner and therefore represent attractive targets for application in molecular plant breeding. We … Transcription factors have been shown to control the activity of multiple stress response genes in a coordinated manner and therefore represent attractive targets for application in molecular plant breeding. We investigated the possibility of modulating the transcriptional regulation of drought and cold responses in the agriculturally important species, wheat and barley, with a view to increase drought and frost tolerance. Transgenic wheat and barley plants were generated showing constitutive (double 35S) and drought-inducible (maize Rab17) expression of the TaDREB2 and TaDREB3 transcription factors isolated from wheat grain. Transgenic populations with constitutive over-expression showed slower growth, delayed flowering and lower grain yields relative to the nontransgenic controls. However, both the TaDREB2 and TaDREB3 transgenic plants showed improved survival under severe drought conditions relative to nontransgenic controls. There were two components to the drought tolerance: real (activation of drought-stress-inducible genes) and 'seeming' (consumption of less water as a result of smaller size and/or slower growth of transgenics compared to controls). The undesired changes in plant development associated with the 'seeming' component of tolerance could be alleviated by using a drought-inducible promoter. In addition to drought tolerance, both TaDREB2 and TaDREB3 transgenic plants with constitutive over-expression of the transgene showed a significant improvement in frost tolerance. The increased expression of TaDREB2 and TaDREB3 lead to elevated expression in the transgenics of 10 other CBF/DREB genes and a large number of stress responsive LEA/COR/DHN genes known to be responsible for the protection of cell from damage and desiccation under stress.

Effect of salinity stress on plants and its tolerance strategies: a review

2014-11-15

Parul Parihar , Samiksha Singh , Rachana Singh +2 more

Genetic approaches to crop improvement: responding to environmental and population changes

2008-05-13

Shin Takeda , Makoto Matsuoka

Agarose Gel Electrophoresis for the Separation of DNA Fragments

2012-04-20

Pei Yun Lee , John Costumbrado , Michael C. Hsu +1 more

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium … Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is "biased reptation", whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 1. Understand the mechanism by which DNA fragments are separated within a gel matrix 2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 3. Identify an agarose solution of appropriate concentration for their needs 4. Prepare an agarose gel for electrophoresis of DNA samples 5. Set up the gel electrophoresis apparatus and power supply 6. Select an appropriate voltage for the separation of DNA fragments 7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 8. Determine the sizes of separated DNA fragments

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Microprep protocol for extraction of DNA from tomato and other herbaceous plants

1995-09-01

T. M. Fulton , Julapark Chunwongse , Steven D. Tanksley

Transformation of intact yeast cells treated with alkali cations

1983-01-01

Hisao Ito , Y Fukuda , Kousaku Murata +1 more

Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A … Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication.

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Dosage-dependent modifiers of polycomb and antennapedia mutations in Drosophila.

1988-11-01

James A. Kennison , John W. Tamkun

Two genes known to control the determination of segmental identity in Drosophila melanogaster are polycomb and antennapedia. To identify additional genes involved in the determination of segmental identity, we have … Two genes known to control the determination of segmental identity in Drosophila melanogaster are polycomb and antennapedia. To identify additional genes involved in the determination of segmental identity, we have isolated dominant modifers (both suppressors and enhancers) of polycomb and/or antennapedia mutations. Sixty-four such modifier mutations have been recovered and mapped to 18 complementation groups. All of the mutations identify genes necessary for viability of the zygote. Six of the 18 genes that were identified by mutations that interact with polycomb and/or antennapedia have been previously characterized as homoeotic genes [i.e., Sex combs reduced (Scr), Brista (Ba), trithorax (trx), Polycomb (Pc), Polycomblike (Pcl), and Sex comb on midleg (Scm)]. Mutations in several of the additional loci identified here have also been shown to have homoeotic phenotypes.

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A gene complex controlling segmentation in Drosophila

1978-12-01

E. B. Lewis

VARIATION IN GENOMIC FORM IN PLANTS AND ITS ECOLOGICAL IMPLICATIONS

1987-05-01

M. D. Bennett

S ummary The gross form of the nuclear genome varies greatly among plant species in both anatomy and genetic organization. Chromosome number (ft) ranges from 2 to over 600, and … S ummary The gross form of the nuclear genome varies greatly among plant species in both anatomy and genetic organization. Chromosome number (ft) ranges from 2 to over 600, and ploidy from 1 to over 20. The amount of DNA in the unreplicated haplophase genome (the 1C value) differs by more than 2500‐fold among angiosperms. Although it has been questioned since the 1930s whether such variation is of adaptive significance and whether it is related, perhaps causally, with environmental factors, no direct or causal links have yet been found. However, variation in DNA C‐value has far‐reaching biological consequences and can be of considerable adaptive and hence ecological significance. Strikingly precise interspecific relationships exist between DNA C‐value and many diverse phenotypic characters at the cellular level, and DNA can affect the phenotype in two ways, firstly by expression of its genie content and, secondly, by the biophysical effects of its mass and volume, the latter defined as nucleotypic effects. Nucleotypic variation in DNA C‐value sets absolute limits to both the minimum size and mass of the basic unit of plant anatomy (i.e. the cell) and the minumum time needed to produce a similar cell with newly synthesized organic molecules. Moreover, in complex multicellular vascular plants, such effects at successive cell cycles are additive, so that DNA C‐value influences many characters, including growth rate, seed weight, minimum generation time and type of life‐cycle. Thus, the nucleotype profoundly affects where, when and how plants grow. Selection for a particular genomic form acting on its spatial or temporal consequences may occur at various levels ranging from the cell to the whole organism and may operate throughout the life‐cycle or at just one stage. DNA C‐value is often indirectly related to environmental factors which determine time‐limited environments via selection acting on the temporal phenotypic consequences of nucleotypic variation. However, in the case of radio‐sensitivity, selection for a low DNA C‐value may act directly on the nucleotype itself, as the size of the nuclear DNA target directly affects the ability of the plant to survive.

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Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

1984-05-01

David C. Schwartz , Charles R. Cantor

Transgenic approaches for abiotic stress tolerance in plants: retrospect and prospects

2007-11-19

Pooja Bhatnagar‐Mathur , Vincent Vadez , Kiran K. Sharma

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Fine Structure Genetic Analysis of a β-Globin Promoter

1986-05-02

R Myers , Kit Tilly , Tom Maniatis

A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse β-major globin gene. The effects … A novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse β-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated β-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription. In contrast, two different mutations in nucleotides immediately upstream from the CCAAT box resulted in a 3- to 3.5-fold increase in transcription. With two minor exceptions, single base substitutions in all other regions of the promoter had no effect on transcription. These results precisely delineate the cis -acting sequences required for accurate and efficient initiation of β-globin transcription, and they establish a general approach for the fine structure genetic analysis of eukaryotic regulatory sequences.

Breeding Technologies to Increase Crop Production in a Changing World

2010-02-11

Mark Tester , Peter Langridge

To feed the several billion people living on this planet, the production of high-quality food must increase with reduced inputs, but this accomplishment will be particularly challenging in the face … To feed the several billion people living on this planet, the production of high-quality food must increase with reduced inputs, but this accomplishment will be particularly challenging in the face of global environmental change. Plant breeders need to focus on traits with the greatest potential to increase yield. Hence, new technologies must be developed to accelerate breeding through improving genotyping and phenotyping methods and by increasing the available genetic diversity in breeding germplasm. The most gain will come from delivering these technologies in developing countries, but the technologies will have to be economically accessible and readily disseminated. Crop improvement through breeding brings immense value relative to investment and offers an effective approach to improving food security.

The origin and behavior of mutable loci in maize

1950-06-01

Barbara McClintock

Metacognition, the ability to reflect upon, and evaluate our own beliefs, can help us avoid making decisions based on unreliable evidence. Here, we provide empirical tests of the importance of … Metacognition, the ability to reflect upon, and evaluate our own beliefs, can help us avoid making decisions based on unreliable evidence. Here, we provide empirical tests of the importance of human metacognition during the COVID-19 pandemic. ...Metacognition, our ability to reflect on our own beliefs, manifests itself in the confidence we have in these beliefs, and helps us guide our behavior in complex and uncertain environments. Here, we provide empirical tests of the importance of ...

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High resolution acrylamide gel electrophoresis of histones

1969-01-01

Sakol Panyim , Roger Chalkley

CHROMOSIN, A DESOXYRIBOSE NUCLEOPROTEIN COMPLEX OF THE CELL NUCLEUS

1946-11-20

A. E. Mirsky , Arthur W. Pollister

A desoxyribose nucleoprotein complex, which we have referred to as a chromosin, has been prepared from a great variety of cells, mainly animal but also plant and bacterial. A chromosin … A desoxyribose nucleoprotein complex, which we have referred to as a chromosin, has been prepared from a great variety of cells, mainly animal but also plant and bacterial. A chromosin is derived from the cell nucleus. In the course of preparation precautions have been taken to prevent contamination by cytoplasmic constituents. To assure the nuclear origin of all components of chromosin, nuclei have in several instances been isolated before extraction was begun. Because of the precautions taken, chromosins do not contain detectable quantities of ribose nucleoproteins; but, incidentally, extraction of ribose nucleoproteins, free of desoxyribose compounds, has also been described in this paper. A typical chromosin contains 3 components: desoxyribose nucleic acid, histone, and non-histone protein. The nucleic acid, being highly polymerized, is exceedingly viscous when dissolved and fibrous when precipitated. Histone and non-histone protein differ from each other in a number of ways, of which one of the most definite is that whereas a histone contains no more than traces of tryptophane, the non-histone protein of chromosin contains nearly 1 per cent of tryptophane. In neutral physiological saline both proteins can combine with nucleic acid. With the isolation of chromosins from so many different kinds of cells, it can now be seen that (contrary to the view expressed by Kossel) histones are present in most animal cells and at least in some plant and bacterial cells. Chromosin prepared from the Type III pneumococcus is active in transforming the type of a pneumococcus culture. It has been pointed out that it is not yet known whether or not protein is a necessary constituent of the transforming agent. To extract chromosin from a cell M NaCl is used. When dissolved in M NaCl the nucleic acid and histone components of a chromosin are to a considerable extent dissociated. They are not dissociated when the chromosin is dissolved in 0.02 M NaCl, but in this medium a partial depolymerization of the nucleic acid occurs. A chromosin should certainly not be considered to be a definite chemical compound. It is a complex extracted from chromatin, which is itself a complicated nuclear structure. And in the course of extraction, it need hardly be said, the structure of chromatin has been considerably changed. To avoid complications it has been considered an advantage in this work to begin with isolated nuclei, and it would clearly be a further simplification to begin chemical procedures only after the chromosomes themselves have been isolated. This is now being accomplished, and it is found that the methods described in this paper are of value in learning how the substances present in a chromosin are put together in a chromosome.

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The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence

1979-04-01

Carl Wu , Paul M. Bingham , Kenneth J. Livak +2 more

Unique Mode of GCC Box Recognition by the DNA-binding Domain of Ethylene-responsive Element-binding Factor (ERF Domain) in Plant

1998-10-01

Dongyun Hao , Masaru Ohme‐Takagi , Akinori Sarai

Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some … Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pm range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity. Ethylene-responsive element-binding proteins (EREBPs)have novel DNA-binding domains (ERF domains), which are widely conserved in plants, and interact specifically with sequences containing AGCCGCC motifs (GCC box). Deletion experiments show that some flanking region at the N terminus of the conserved 59-amino acid ERF domain is required for stable binding to the GCC box. Three ERF domain-containing fragments of EREBP2, EREBP4, and AtERF1 from tobacco and Arabidopsis, bind to the sequence containing the GCC box with a high binding affinity in the pm range. The high affinity binding is conferred by a monomeric ERF domain fragment, and DNA truncation experiments show that only 11-base pair DNA containing the GCC box is sufficient for stable ERF domain interaction. Systematic DNA mutation analyses demonstrate that the specific amino acid contacts are confined within the 6-base pair GCCGCC region of the GCC box, and the first G, the fourth G, and the sixth C exhibit highest binding specificity common in all three ERF domain-containing fragments studied. Other bases within the GCC box exhibit modulated binding specificity varying from protein to protein, implying that these positions are important for differential binding by different EREBPs. The conserved N-terminal half is likely responsible for formation of a stable complex with the GCC box and the divergent C-terminal half for modulating the specificity. pathogenesis-related ethylene-responsive element ERE-binding protein ERE-binding factor base pair(s) amino acid(s) electrophoresis mobility shift binding assay N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine polyacrylamide gel electro- phoresis. The plant hormone ethylene regulates many complex processes of stress responses and developmental adaptations in higher plants (1Abeles F.B. Morgan P.W. Saltveit Jr., M.E. Ethylene in Plant Biology. 2nd Ed. Academic Press, Inc., New York1992Google Scholar). Stimulation of expression of pathogenesis-related (PR)1 genes, such as class I basic chitinases and β-1,3-glucanases, is among the consequences of ethylene involvement (2Ohme-Takagi M. Shinshi H. Plant Mol. Biol. 1990; 15: 941-946Crossref PubMed Scopus (105) Google Scholar). Analysis of the promoter regions of ethylene-inducible PR genes has led to the identification of an ethylene-responsive element (ERE) (3Sato F. Kitajima S. Koyama T. Yamada Y. Plant Cell Physiol. 1996; 37: 249-255Crossref PubMed Scopus (81) Google Scholar, 4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar), which contains a conserved AGCCGCC motif (2Ohme-Takagi M. Shinshi H. Plant Mol. Biol. 1990; 15: 941-946Crossref PubMed Scopus (105) Google Scholar, 5Eyal Y. Meller Y. Lev-Yadun S. Fluhr R. Plant J. 1993; 4: 225-234Crossref PubMed Scopus (136) Google Scholar, 6Hart C.M. Nagy F. Meins Jr., F. Plamt Mol. Biol. 1993; 21: 121-131Crossref PubMed Scopus (91) Google Scholar, 7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar) designated as the GCC box or occasionally the PR box (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar, 7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar). Four different cDNAs encoding the GCC box-specific binding factors (EREBPs) have been isolated from tobacco (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar). The deduced amino acid sequences of EREBPs neither show homology to known DNA-binding proteins and transcription factors nor do they contain basic leucine zipper or zinc finger motifs, suggesting that EREBPs are a group of novel DNA-binding proteins. EREBPs contain highly conserved 58–59 sequences of residues (designated as ERF domains in this paper, see Fig. 1) within which GCC box-specific binding activity was identified (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar). Recently, it has been reported that ERF domains exist in numerous products of regulatory genes and cDNAs of unknown function from various plants (7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar, 8Okamuro J.K. Caster B. Villarroel R. Van Montagu M. Jofuku K.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7076-7081Crossref PubMed Scopus (518) Google Scholar), but they are not found in mammalian, fungi, or yeast (9Weigel D. Plant Cell. 1995; 7: 388-389Crossref PubMed Scopus (155) Google Scholar). Whereas ERF domains are highly conserved, EREBPs exhibit some divergence. The ERF domain also has been argued to be closely related to the AP2 domain of Arabidopsis (8Okamuro J.K. Caster B. Villarroel R. Van Montagu M. Jofuku K.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7076-7081Crossref PubMed Scopus (518) Google Scholar, 10Stockinger E.J. Gilmour S.J. Thomashow M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1035-1040Crossref PubMed Scopus (1449) Google Scholar) although the DNA-binding activity of AP2 has not been proven nor has its target sequence been identified. Thus, it has been suggested that these transcription factors belong to two subdivisions (7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar, 9Weigel D. Plant Cell. 1995; 7: 388-389Crossref PubMed Scopus (155) Google Scholar, 11Büttner M. Singh K.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5961-5966Crossref PubMed Scopus (298) Google Scholar), with fewer amino acids being identical in all members of the extended family. Moreover, AP2 contains two DNA-binding domains whereas EREBPs have only one (9Weigel D. Plant Cell. 1995; 7: 388-389Crossref PubMed Scopus (155) Google Scholar). Among the proteins containing the ERF domain, EREBPs (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar), AtEBP (11Büttner M. Singh K.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5961-5966Crossref PubMed Scopus (298) Google Scholar), Pti4–6 (7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar), and AtERFs (Arabidopsis ethylene-responsive transcription factor) 2M. Ohta, M. Ohme-Takagi, and H. Shinshi, unpublished data. have been shown to have GCC box-specific binding activity. However, their DNA recognition mechanism and biological function are not yet understood. It is also unclear whether each ERF domain exhibits the same binding specificity with the GCC box. Attempts in this report were made to establish the GCC box-binding specificity of ERF domain by single-base substitution of the GCC box and quantitative electrophoretic mobility shift assay (EMSA). The GCC box binding affinities of three ERF domain-containing fragments, from tobacco (EREBP2 and EREBP4) and Arabidopsis (AtERF1), were examined, and the stoichiometry of their DNA interactions were established. Truncation analyses of the ERF domain fragment have revealed a minimal region for stable GCC box binding, and systematic mutation and truncation assays identified crucial bases within the GCC box responsible for specific protein-DNA interaction. The coding region of ERF domain fragments from EREBP2 (aa 88–164), EREBP4 (aa 122–222), and AtERF1 (aa 138–213), designated as EBP2-F, EBP4-F, and AtERF1-F, respectively, in this paper, were prepared by PCR, cloned into pET3C plasmid (Novagen), and expressed in BL21(DE3)pLysS Escherichia coli cells. After freeze-thaw and subsequent gentle sonication, the cell lysate was clarified and applied to heparin columns (Pros HE, PerSeptive Biosystems). Fractions containing ERF domain fragments were filtrated through Superdex 75 (Amersham Pharmacia Biotech) and further purified by cation exchange chromatography (Resource S, Amersham Pharmacia Biotech). The purified ERF domain fragments were concentrated and dialyzed against water. For preparation of the fusion proteins with maltose-binding protein, the coding region of EBP4-F or tobacco TGA1a (12Katagiri F. Lam E. Chua N.H. Nature. 1989; 340: 727-730Crossref PubMed Scopus (334) Google Scholar) were cloned into pMAL-2 plasmid (New England Biolabs Inc.), expressed in BL21 E. coli cells, and purified using amylose columns according to manufacturer instructions. Protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent kit (Pierce) and further confirmed by the methods of Gill and von Hippel (13Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5083) Google Scholar). A 16-bp GCC box fragment (5′-CATAAGAGCCGCCACT-3′) of the 5′ upstream region of the tobacco Gln2 gene was prepared by synthesizing both strands (2Ohme-Takagi M. Shinshi H. Plant Mol. Biol. 1990; 15: 941-946Crossref PubMed Scopus (105) Google Scholar). After purification, the fragments were suspended in 1 × STE buffer, and the complementary strands were annealed. The resulting double strand oligonucleotides were labeled with [γ-32P]ATP (NEN Life Science Products) by T4 polynucleotide kinase (Toyobo) and purified through a Sephadex G-50 column before binding experiments. Mutant and truncated sequences (see Table I) were made in the same way.Table ITruncational and mutational binding assays for GCC boxnameGCC box segquencesKdWt16CATAAGAGCCGCCACT70 ± 58 pmMmgtattcAGCCGCCtga52 ± 17 pmMdCATAAGAtCCtCCACT>1.0 μmT14TAAGAGCCGCCACT44 ± 17 pmT12AGAGCCGCCACT81 ± 20 pmT12aGAGCCGCCACTG71 ± 30 pmT12bAGCCGCCACTGA>1.0 μmT12cAAGAGCCGCCAC>1.0 μmT12dTAAGAGCCGCCA>1.0 μmT11GAGCCGCCACT73 ± 28 pmA 16-mer wild type GCC box sequence (Wt16) contains a core region of -AGCCGCC- (highlighted and underlined). Quantitative EMSA was performed by using Wt16 or different mutants (M) and truncates (T) and EBP2-F at various concentrations. The numbers after T indicate the length of the oligos. The data for K d values are represented as the mean of three replicates ± S.D. K d values for nonspecific binding is about 0.1–1.0 μm or higher. Open table in a new tab A 16-mer wild type GCC box sequence (Wt16) contains a core region of -AGCCGCC- (highlighted and underlined). Quantitative EMSA was performed by using Wt16 or different mutants (M) and truncates (T) and EBP2-F at various concentrations. The numbers after T indicate the length of the oligos. The data for K d values are represented as the mean of three replicates ± S.D. K d values for nonspecific binding is about 0.1–1.0 μm or higher. Equilibrium dissociation constants (K d) of ERF domain-DNA binding were measured by quantitative EMSA. Labeled DNA (at a concentration of 10–100-fold lower than the K d) was mixed with a gradient concentration of ERF domain fragments in 10 μl of binding buffer (25 mm HEPES-KOH buffer at pH 7.5, containing 40 mmKCl, 0.1 mm EDTA, 0.1 mg/ml bovine serum albumin, and 10% glycerol). After incubation at room temperature for 15 min, the samples were run on an 8% nondenaturing PAGE, and the resulting gel was dried and visualized on a FUJIX BAS-2000 system (Fuji). Complexed and free DNA bands were scanned, their photodensity was measured, and K d was determined from binding titration analysis. The binding free energy change (ΔΔG) was calculated as: ΔΔG = RT ln [K d(mutant)/K d (wild-type)], where R is the gas constant and T is absolute temperature. Positive ΔΔG represents a decreased binding affinity, negative ΔΔG corresponds to an increased binding affinity, and zero ΔΔG means no effect on binding. A 10-fold decrease in binding affinity increases ΔΔG by about 1.3 kcal/mol. For protein cross-linking experiments, the purified proteins were incubated with glutaraldehyde in total volume of 4 μl in DNA-binding buffer (see above). After a 10-min incubation at room temperature, cross-linked products were separated by standard Tris-glycine, pH 8.3, discontinuous SDS-PAGE, or for smaller polypeptide, in the Tris-Tricine system (14Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10527) Google Scholar). Derivatives of a fragment of EREBP2 (EBP2-F), containing an ERF domain and 10-aa N-terminal and 8-aa C-terminal flanking regions, were used to identify the minimal sequence for GCC box binding. The different truncates of this fragment were subjected to EMSA for examination of the binding activity to the 16-bp GCC box sequence (Wt16, see Table I). Fig. 2 shows that EBP2-F exhibits a high binding activity to the GCC box. Deletion of 10 aa from the N terminus (Ec, aa 98–164) or both N- and C-terminal flanking regions (Enc, aa 98–156) leads to either a dramatic decrease or complete abolition of binding activity with the GCC box. Elimination of the C-terminal flanking region (En, aa 88–156) causes only a negligible decrease in binding affinity in comparison with EBP2-F. This indicates that the short stretch of N-terminal flanking region is necessary for binding of the ERF domains to the GCC box. To measure the equilibrium binding of the EREBPs, labeled wild type 16-bp GCC box sequence was subjected to EMSA for interaction with EBP2-F, EBP4-F, or AtERF1-F. Upon titration analysis, the equilibrium dissociation constant (K d) was determined under the given binding condition. All three fragments exhibit a high binding affinity with K d in a range from about 1 to 120 pm. The highest binding was found with EBP4-F (K d = 5.4 ± 4.3 pm) and the lowest was with AtERF1-F (K d = 167 ± 40 pm), whereas the K d for EBP2-F was intermediate at about 70 ± 58 pm. A similarK d was also observed for the binding between full-length EREBP2 and the GCC box. The high binding activity observed above led to consideration of whether a dimer or even oligomers of ERF domain fragments form in free solution or under DNA-binding condition. The possibility of dimerization of ERF domain was investigated by EMSA using two ERF domain fragments of different sizes. A maltose-binding protein fused to EBP4-F (Mal-EBP4-F, 53 kDa) was mixed with EBP4-F (11 kDa) in varying concentration before binding to the GCC box. The results shown in Fig. 3 illustrate that a mixture of these two molecules (lanes 8–10) at equivalent concentrations shift the DNA into two bands corresponding to the individual proteins (lanes 2–7), and no extra band was observed. When the Mal-EBP4-F concentration is kept constant at 1.0 nm while varying EBP4-F from 10 pm to 0.1 μm(lane 11–13), the DNA shifts reflect an intercompetitive behavior of the proteins. These results indicate the absence of an hetero-complex under the given binding condition. To further examine the possibility of an intrinsic homo-dimer present in binding buffer or DNA-induced dimerization, EBP2-F with or without interaction of the GCC box was cross-linked with increasing concentrations of glutaraldehyde and then subjected to standard SDS-PAGE. In a control experiment (Fig. 4 A), the bZip protein of tobacco TGA1a, known to be a dimer in solution (12Katagiri F. Lam E. Chua N.H. Nature. 1989; 340: 727-730Crossref PubMed Scopus (334) Google Scholar), was examined to confirm that glutaraldehyde cross-linking can be used to detect oligomerization. Using DNA-binding conditions in the presence or absence of the GCC box, no bands corresponding to higher forms of EBP2-F (8.3 kDa) were observed regardless of the concentration of glutaraldehyde or the presence of GCC box sequence (Fig. 4, B and C). This suggests that EBP2-F exists in binding solution as a monomer and that no dimerization is induced upon binding with GCC box. To determine the minimal GCC box motif required for stable binding by EREBPs, truncations were made at the surroundings of the GCC box (underlined) as presented in Table I, and the binding assays with EBP2-F were performed. Table I shows that the 16-bp GCC box can be shortened by up to 5 bp (T14, T12 and T12a) at the 5′ terminus without effect on binding affinity. Shortening the 3′ terminus destroys the binding completely (T12c and T12d). This indicates that the 11-bp DNA fragment (T11) is the minimal GCC box sequence bound by ERF domain fragments and that flanking sequences 1 bp at 5′ and 3 bp at 3′ terminus outside of the GCC box are necessary for the stable binding of EREBPs. Table I also shows that alteration of the entire surrounding region of the GCC box at both the 5′ and 3′ termini (Mm) has no effect on binding affinity. However, double substitution of G with T at positions 8 and 11 (Md) within the GCC box eliminates the binding. This suggests that the GCC box,i.e. AGCCGCC, is primarily responsible for specific protein binding. To further detail the ERF domain binding specificity of each individual base pair within the GCC box, EMSA with the three ERF domain fragments was performed with GCC box mutants possessing systematic single-base substitutions. Each base in the GCC box from A7 to C13 was substituted with the other three (for sequence numbering, see Fig. 5), and free energy changes (ΔΔG) were obtained from quantitative titration analysis. The results presented in Fig. 5 show that for all three ERF domain fragments, base substitutions at G8, G11, and C13, cause the greatest decrease in binding affinity, indicating that these three positions involve highly specific interactions with EREBPs. The importance of G8 and G11 in maintaining the binding specificity with EREBPs agrees with other reports (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar, 7Zhou J. Tang X. Martin G.B. EMBO J. 1997; 16: 3217-3218Google Scholar), but C13 also appears to be an important base within the GCC box. For all three ERF domain fragments tested, substitution of A at position 7 of the GCC box with other bases shows little effect on binding except for AtERF1-F, whose conversion of A7 to T7 causes an ∼10-fold decrease in binding affinity. In addition, the ERF domain fragments are differentially affected by base substitutions at positions other than G8, G11, and C13. For example, substitutions of C9 to G and C10 to T destroy EBP2-F binding completely, whereas substitution of C12 to either G or T has little effect on the binding affinity of EBP4-F. Therefore, specific contacts may occur between the EREBPs and the GCCGCC region of the GCC box, but modulated interactions may also occur with the various proteins of the ERF family. Studies with truncations of EBP2-F (see Fig. 2) have revealed that its central region, which includes the 59-aa ERF domain plus a short N-terminal stretch, is necessary for GCC box binding. This is consistent with a similar observation of Ohme-Takagi and Shinshi (4Ohme-Takagi M. Shinshi H. Plant Cell. 1995; 7: 173-182Crossref PubMed Scopus (946) Google Scholar). However, the ERF domain itself does not possess any binding activity, and the N-terminal flanking appears to be crucial for binding with the GCC box. It is unclear if the N-terminal amino acids specifically interact with the GCC box. Because there is no significant homology at N-terminal flanking region among the family of ERF domain-containing proteins, it may not be responsible for the specific GCC box interactions, but instead maintains an appropriate conformation of the ERF domain for DNA binding. The present study also shows that ERF domain fragments from tobacco and Arabidopsis bind to 16-bp oligonucleotides containing the GCC box from the 5′ upstream region of the tobacco PR geneGln2. All three fragments studied exhibit high binding affinity to the GCC box with K d in the pm range. Such a high binding constant is similar to that of prokaryotic repressors, such as the Cro/OR1 operator (K d = 4.0 pm, Ref. 15Takeda Y. Sarai A. Rivera V.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 439-443Crossref PubMed Scopus (174) Google Scholar) and lac/operator (K d = 0.1 pm, Ref. 16Riggs A.D. Suzuki H. Bourgeois S. J. Mol. Biol. 1970; 48: 67-83Crossref PubMed Scopus (549) Google Scholar), and eukaryotic transcription factors such as c-Myb/enhancer (K d = 0.4 nm, Ref. 17Tanikawa J. Yasukawa T. Enari M. Ogata K. Nishimura Y Ishii S. Sarai A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9320-9324Crossref PubMed Scopus (135) Google Scholar). All of the latter DNA-binding proteins are either dimers or contain homologous tandem repeats, interacting with their DNA targets of 16/17-bp in a cooperative manner. However, gel shift binding assays for dimer formation revealed that each ERF domain fragments interact as independent units with the GCC box (see Fig. 3). Indeed, the results from protein cross-linking experiments (see Fig. 4) and native gel filtration of EBP2-F (data not shown) strongly suggest that the ERF domain fragment is a monomer in solution, and the GCC box does not induce dimerization upon protein binding. Furthermore, truncation assays (see Table I) reveal that the 11-bp GCC box plus 1 bp at the 5′ and 3 bp at the 3′ flanking regions are sufficient for the binding activity of EREBPs. To our knowledge, this high-affinity binding between a monomeric protein domain and such a short DNA sequence has not been reported. High affinity may be facilitated by the abundant G-C base pairs in the GCC box, the positive charge of ERF domain fragments (average pI = 10.2), and/or enhancement of intermolecular contact through conformational rearrangements of the ERF domains or DNA deformation such as DNA bending. In any case, the present results suggest that a novel mode of DNA binding is involved in interactions of ERF domains with the GCC box. Quantitative EMSA with systematic single-base substitutions of the 7-bp GCC box reveal that the strongest contacts with the ERF domain fragments occur at G8, G11, and C13, whereas A7 is minimally involved. Sequence comparison (see Fig. 1) of the conserved 59-aa ERF domain among the three ERF domain fragments studied shows that their N-terminal halves are almost identical. This region appears to coincide with the GCC box binding specificity of all three ERF domain fragments observed in this study, implying that the N-terminal half is responsible for the specific recognition of the GCC box. Okamuro et al. (8Okamuro J.K. Caster B. Villarroel R. Van Montagu M. Jofuku K.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7076-7081Crossref PubMed Scopus (518) Google Scholar) speculated that an N-terminal region (designated the YRG element) of EREBP-like proteins, which may be shorter than what we refer here, may function in DNA binding because of the abundance of basic amino acids at this conserved region. In addition, many proteins (those marked with an asterisk in Fig. 1) with similar N-terminal half in their ERF domain have been shown to possess GCC box binding activity. On the other hand, TINY and CBF1 (see Fig. 1) have much less sequence homology in their ERF domains, suggesting that they recognize a distinct target site. In fact, CBF1 has been shown to bind a 5-bp sequence of CCGAC (10Stockinger E.J. Gilmour S.J. Thomashow M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1035-1040Crossref PubMed Scopus (1449) Google Scholar) rather than the 6-bp GCCGCC. The ΔΔGs obtained from the systematic mutation analysis show some variations of GCC box binding specificity at the positions such as C9, C10, and C12 among the three ERF domain fragments (see Fig. 5). This is probably related to divergence of the sequences of the C-terminal halves of the three ERF domain fragments studied, a region with high amphipathic α-helix propensity. Although this region might not interact with the GCC box directly, its sequence diversity may cause conformational difference of the ERF domains, indirectly affecting the binding specificity with the GCC box. The amphipathic α-helix might also interact with other regulatory factors because this region is common in the conserved ERF domain and often mediates protein-protein interactions (9Weigel D. Plant Cell. 1995; 7: 388-389Crossref PubMed Scopus (155) Google Scholar, 18Jofuku K.D. den Boer B.G.W. Van Montagu M. Okamuro J.K. Plant Cell. 1994; 6: 1211-1225Crossref PubMed Scopus (852) Google Scholar). The differences in GCC box binding specificity found in this study therefore may play important roles in regulating the expression of appropriate PR genes by different ERF domain-containing proteins. We thank Dr. Yuan Chang for contribution at the initial stage of this work.

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Discovery of chemically induced mutations in rice by TILLING

2007-04-11

Bradley J. Till , Jennifer L. Cooper , Thomas H. Tai +4 more

Abstract Background Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic … Abstract Background Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method. Results To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7–1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population. Conclusion We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.

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HISTONE, A SUPPRESSOR OF CHROMOSOMAL RNA SYNTHESIS

1962-07-01

Ru-chih C. Huang , James Bonner

We have previously reported(1,2) that chromatin isolated from pea embryos possesses the ability to carry out the DNA-dependent synthesis of RNA from the four riboside triphosphates.(3) The present paper concerns … We have previously reported(1,2) that chromatin isolated from pea embryos possesses the ability to carry out the DNA-dependent synthesis of RNA from the four riboside triphosphates.(3) The present paper concerns the roles in such synthesis of the several components of chromatin. It will be shown that the DNA of pea embryo chromatin is present in at least two forms, namely, as DNA itself and as DNA bound in nucleohistone complex. It will be further shown that DNA fully complexed with histone is inactive in the support of DNA-dependent RNA synthesis.

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The c-mos proto-oncogene product is a cytostatic factor responsible for meiotic arrest in vertebrate eggs

1989-11-01

Noriyuki Sagata , Nobumoto Watanabe , George F. Vande Woude +1 more

A small cosmid for efficient cloning of large DNA fragments

1980-11-01

Barbara Höhn , John Collins

Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice

2012-11-06

Satoru Ishikawa , Yasuhiro Ishimaru , Masato Igura +7 more

Rice ( Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon … Rice ( Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd⋅kg −1 in the grain compared with a mean of 1.73 mg Cd⋅kg −1 in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene ( OsNRAMP5 ), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5 . When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

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Controlling Elements and the Gene

1956-01-01

Barbara McClintock

In a recent brief review (McClintock, 1956), a description was given of types of elements carried in the maize chromosomes that serve to control gene action and to induce, at … In a recent brief review (McClintock, 1956), a description was given of types of elements carried in the maize chromosomes that serve to control gene action and to induce, at the site of the gene, heritable modifications affecting this action. These elements were initially discovered because they do not remain at one position in the chromosome complement. They can appear at new locations and disappear from previously determined locations. The presence of one such element at or near the locus of a known gene may affect the action of this gene. In so doing, it need not alter the action potentials of the genic substances at the locus. Therefore, these elements were called controlling elements. It was also shown that controlling elements fall into groups, the members of each operating as an integrated system in the control of gene action.

Genetic Engineering for Modern Agriculture: Challenges and Perspectives

2010-05-04

Ron Mittler , Eduardo Blumwald

Abiotic stress conditions such as drought, heat, or salinity cause extensive losses to agricultural production worldwide. Progress in generating transgenic crops with enhanced tolerance to abiotic stresses has nevertheless been … Abiotic stress conditions such as drought, heat, or salinity cause extensive losses to agricultural production worldwide. Progress in generating transgenic crops with enhanced tolerance to abiotic stresses has nevertheless been slow. The complex field environment with its heterogenic conditions, abiotic stress combinations, and global climatic changes are but a few of the challenges facing modern agriculture. A combination of approaches will likely be needed to significantly improve the abiotic stress tolerance of crops in the field. These will include mechanistic understanding and subsequent utilization of stress response and stress acclimation networks, with careful attention to field growth conditions, extensive testing in the laboratory, greenhouse, and the field; the use of innovative approaches that take into consideration the genetic background and physiology of different crops; the use of enzymes and proteins from other organisms; and the integration of QTL mapping and other genetic and breeding tools.

THE DNA OF CAENORHABDITIS ELEGANS

1974-05-01

John Sulston , Sydney Brenner

ABSTRACT Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x IO7 base pairs (20 times the genome … ABSTRACT Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x IO7 base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4s RNA, 110 for 5s RNA, and 55 for (18 + 28)S RNA.

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Genomics-based approaches to improve drought tolerance of crops

2006-07-26

Roberto Tuberosa , Silvio Salvi

Nuclear DNA content and minimum generation time in herbaceous plants

1972-06-06

M. D. Bennett

Many components of cell and nuclear size and mass are correlated with nuclear DNA content in plants, as also are the durations and rates of such developmental processes as mitosis … Many components of cell and nuclear size and mass are correlated with nuclear DNA content in plants, as also are the durations and rates of such developmental processes as mitosis and meiosis. It is suggested that the multiple effects of the mass of nuclear DNA which affect all cells and apply throughout the life of the plant can together determine the minimum generation time for each species. The durations of mitosis and of meiosis are both positively correlated with nuclear DNA content and, therefore, species with a short minimum generation time might be expected to have a shorter mean cell cycle time and mean meiotic duration, and a lower mean nuclear DNA content, than species with a long mean minimum generation time. In tests of this hypothesis, using data collated from the literature, it is shown that the mean cell cycle time and the mean meiotic duration in annual species is significantly shorter than in perennial species. Furthermore, the mean nuclear DNA content of annual species is significantly lower than for perennial species both in dicotyledons and monocotyledons. Ephemeral species have a significantly lower mean nuclear DNA content than annual species. Among perennial monocotyledons the mean nuclear DNA content of species which can complete a life cycle within one year (facultative perennials) is significantly lower than the mean nuclear DNA content of those which cannot (obligate perennials). However, the mean nuclear DNA content of facultative perennials does not differ significantly from the mean for annual species. It is suggested that the effects of nuclear DNA content on the duration of developmental processes are most obvious during its determinant stages, and that the largest effects of nuclear DNA mass are expressed at times when development is slowest, for instance, during meiosis or at low temperature. It has been suggested that DNA influences development in two ways, directly through its informational content, and indirectly by the physical-mechanical effects of its mass. The term 'nucleotype' is used to describe those conditions of the nucleus which effect the phenotype independently of the informational content of the DNA. It is suggested that cell cycle time, meiotic duration, and minimum generation time are determined by the nucleotype. In addition, it may be that satellite DNA is significant in its nucleotypic effects on developmental processes.

TargetingInducedLocalLesionsIN Genomes (TILLING) for Plant Functional Genomics

2000-06-01

Claire M. McCallum , Luca Comai , Elizabeth A. Greene +1 more

One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced ([Muller, 1930][1]; [Stadler, 1932][2]). The high frequency with which ionizing radiation … One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced ([Muller, 1930][1]; [Stadler, 1932][2]). The high frequency with which ionizing radiation and certain chemicals can cause genes to mutate made it possible to perform genetic studies

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Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING

2003-03-01

Bradley J. Till , Steven H. Reynolds , Elizabeth A. Greene +7 more

TILLING ( T argeting I nduced L ocal L esions in G enomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of … TILLING ( T argeting I nduced L ocal L esions in G enomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

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Global impact of mutation-derived varieties

2004-01-01

B. S. Ahloowalia , M. Małuszyński , K. Nichterlein

Discovery of induced point mutations in maize genes by TILLING.

2004-07-28

Bradley J. Till , Steven H. Reynolds , Clifford F. Weil +7 more

Abstract Background Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics … Abstract Background Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community. Results We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious. Conclusions Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.

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Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice

2006-01-11

Toshitsugu Nakano , Kaoru Suzuki , Tatsuhito Fujimura +1 more

Abstract Genes in the ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. In this study, a comprehensive computational analysis … Abstract Genes in the ERF family encode transcriptional regulators with a variety of functions involved in the developmental and physiological processes in plants. In this study, a comprehensive computational analysis identified 122 and 139 ERF family genes in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L. subsp. japonica), respectively. A complete overview of this gene family in Arabidopsis is presented, including the gene structures, phylogeny, chromosome locations, and conserved motifs. In addition, a comparative analysis between these genes in Arabidopsis and rice was performed. As a result of these analyses, the ERF families in Arabidopsis and rice were divided into 12 and 15 groups, respectively, and several of these groups were further divided into subgroups. Based on the observation that 11 of these groups were present in both Arabidopsis and rice, it was concluded that the major functional diversification within the ERF family predated the monocot/dicot divergence. In contrast, some groups/subgroups are species specific. We discuss the relationship between the structure and function of the ERF family proteins based on these results and published information. It was further concluded that the expansion of the ERF family in plants might have been due to chromosomal/segmental duplication and tandem duplication, as well as more ancient transposition and homing. These results will be useful for future functional analyses of the ERF family genes.

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Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement

2008-07-01

Stephen P. Moose , Rita H. Mumm

The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in … The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding

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Spectrum of Chemically Induced Mutations From a Large-Scale Reverse-Genetic Screen in Arabidopsis

2003-06-01

Elizabeth A. Greene , Christine A. Codomo , Nicholas E. Taylor +7 more

Abstract Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens … Abstract Chemical mutagenesis has been the workhorse of traditional genetics, but it has not been possible to determine underlying rates or distributions of mutations from phenotypic screens. However, reverse-genetic screens can be used to provide an unbiased ascertainment of mutation statistics. Here we report a comprehensive analysis of ∼1900 ethyl methanesulfonate (EMS)-induced mutations in 192 Arabidopsis thaliana target genes from a large-scale TILLING reverse-genetic project, about two orders of magnitude larger than previous such efforts. From this large data set, we are able to draw strong inferences about the occurrence and randomness of chemically induced mutations. We provide evidence that we have detected the large majority of mutations in the regions screened and confirm the robustness of the high-throughput TILLING method; therefore, any deviations from randomness can be attributed to selectional or mutational biases. Overall, we detect twice as many heterozygotes as homozygotes, as expected; however, for mutations that are predicted to truncate an encoded protein, we detect a ratio of 3.6:1, indicating selection against homozygous deleterious mutations. As expected for alkylation of guanine by EMS, &gt;99% of mutations are G/C-to-A/T transitions. A nearest-neighbor bias around the mutated base pair suggests that mismatch repair counteracts alkylation damage.

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Nuclear DNA amounts in angiosperms

1982-09-22

M. D. Bennett , James B. Smith , J. S. Heslop‐Harrison

Nuclear DNA amounts have been estimated for more than 200 angiosperm species since the last collected list of such values for about 750 species was published by Bennett & Smith … Nuclear DNA amounts have been estimated for more than 200 angiosperm species since the last collected list of such values for about 750 species was published by Bennett & Smith in 1976 ( Phil. Trans. R. Soc. Lond. B 274, 227- 274). These new estimates are either scattered in a wide range of scientific journals or, in many cases, unpublished; so they are not readily accessible. A publication, collecting these data in a single list is required. This paper contains a supplementary list of absolute DNA values, including estimates for 240 angiosperm species not listed by Bennett & Smith in 1976, as well as additional estimates for 41 species already listed by them. These data are assembled primarily for reference purposes. Consequently, the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists, who it is anticipated will be among the major users.

Transgeneration memory of stress in plants

2006-08-01

Jean Molinier , Gerhard Ries , Cyril Zipfel +1 more

Improving the protein content and composition of cereal grain

2007-06-26

Peter R. Shewry

High-Throughput Screening for Induced Point Mutations

2001-06-01

Trenton Colbert , Bradley J. Till , Rachel Tompa +6 more

With the completion of genome sequencing projects, emphasis in genomics has shifted from analyzing sequences to understanding gene function, and effective reverse genetic strategies are increasingly in demand. Here we … With the completion of genome sequencing projects, emphasis in genomics has shifted from analyzing sequences to understanding gene function, and effective reverse genetic strategies are increasingly in demand. Here we report adaptations of the targeting induced local lesions in genomes (TILLING)

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Nuclear DNA amounts in angiosperms

1976-05-27

M. D. Bennett , James B. Smith

The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication … The number of angiosperm species for which nuclear DNA amount estimates have been made has nearly trebled since the last collected lists of such values were published, and therefore, publication of a more comprehensive list is overdue. This paper lists absolute nuclear DNA amounts for 753 angiosperm species. The data were assembled primarily for reference purposes, and so the species are listed in alphabetical order, as this was felt to be more helpful to cyto- and biochemists whom, it is anticipated, will be among its major users. The paper also reviews aspects of the history, nomenclature, methods, accuracy and problems of nuclear DNA estimation in angiosperms. No attempt is made to reconsider those aspects of nuclear DNA estimation which have been fully revised previously, although the bibliography of such aspects is given. Instead, the paper is intended as a source of basic information regarding the terminology, practice and limitations of nuclear DNA estimation, especially by Feulgen microdensitometry, as currently practiced.

Principle and application of plant mutagenesis in crop improvement: a review

2015-12-14

Yusuff Oladosu , Mohd Y. Rafii , Norhani Abdullah +5 more

The first step in plant breeding is to identify suitable genotypes containing the desired genes among existing varieties, or to create one if it is not found in nature. In … The first step in plant breeding is to identify suitable genotypes containing the desired genes among existing varieties, or to create one if it is not found in nature. In nature, variation occurs mainly as a result of mutations and without it, plant breeding would be impossible. In this context, the major aim in mutation-based breeding is to develop and improve well-adapted plant varieties by modifying one or two major traits to increase their productivity or quality. Both physical and chemical mutagenesis is used in inducing mutations in seeds and other planting materials. Then, selection for agronomic traits is done in the first generation, whereby most mutant lines may be discarded. The agronomic traits are confirmed in the second and third generations through evident phenotypic stability, while other evaluations are carried out in the subsequent generations. Finally, only the mutant lines with desirable traits are selected as a new variety or as a parent line for cross breeding. New varieties derived by induced mutatgenesis are used worldwide: rice in Vietnam, Thailand, China and the United States; durum wheat in Italy and Bulgaria; barley in Peru and European nations; soybean in Vietnam and China; wheat in China; as well as leguminous food crops in Pakistan and India. This paper integrates available data about the impact of mutation breeding-derived crop varieties around the world and highlights the potential of mutation breeding as a flexible and practicable approach applicable to any crop provided that appropriate objectives and selection methods are used.

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Advances in Molecular Breeding Toward Drought and Salt Tolerant Crops

2007-01-01

Matthew A. Jenks , Paul M. Hasegawa , S. Mohan Jain

Breeding crops to feed 10 billion

2019-06-17

Lee T. Hickey , Amber N. Hafeez , Hannah Robinson +7 more

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Global Impact of Salinity and Agricultural Ecosystems

2006-03-29

Michael G. Pitman , André Läuchli

The AT-rich regions correspond to the localized centromeres in meiotic chromosomes of Drosera filiformis Raf.

2025-06-24

Yoshikazu Hoshi , K. Terasaki | CYTOLOGIA

Role of Marker-Assisted Breeding in Crop Improvement

2025-06-24

Shubham Sharma , Jatin Sharma , Inderjit Singh +1 more | CRC Press eBooks

Identification of Morphological Agronomic and Quality Characteristics of Hungarian Vetch (Vicia pannonica Crantz.) Mutants

2025-06-23

Berna Efe , Sabahaddin Ünal , Hacer Mintaş +2 more | Turkish Journal Of Field Crops

Gamma-ray irradiation of seeds is an effective method for creating genetic variation in plant breeding. The objective of this study was to evaluate the morphological, agronomic, and quality characteristics of … Gamma-ray irradiation of seeds is an effective method for creating genetic variation in plant breeding. The objective of this study was to evaluate the morphological, agronomic, and quality characteristics of Hungarian vetch (Vicia pannonica Crantz.) mutant lines that were obtained through gamma radiation. The experiment was carried out in Gölbaşı-Ankara during the 2019 and 2020 growing seasons according to randomized complete block design with four replications, involving eleven mutant lines and two control cultivars. The mutant lines exhibited a range of outcomes, including both positive and negative effects. However, no significant alterations were observed in the quality characteristics. The TB1007 mutant line demonstrated superior yield and quality, whereas the TB1002 and TB1005 mutant lines only performed well in terms of yield. The highest dry yield were obtained from TB1007 and TB1002, with 425.0 and 419.3 kg da-1, respectively, followed by the cv. Tarm Beyazı-98 and TB1005 mutant, with 391.4 and 390.1 kg da-1, respectively, in 2019. The TB1007 mutant line had the highest amount of digestible protein, with a content of 16.4% in 2019 and 16.7% in 2020, averaging at 16.5%. These lines are suitable for breeding programmes, especially for future studies involving crosses.

Genome-Wide Identification of the DOG1 Gene Family in Pepper (Capsicum annuum) and Its Expression Profiles During Seed Germination

2025-06-22

Zhichao Zhao , Jingbo Sun , Feng Zhang +1 more | Plants

The DOG1 (Delay of Germination1) family plays key regulatory roles in seed dormancy and germination. However, a genome-wide analysis of DOG1 genes has not been performed for pepper (Capsicum annuum), … The DOG1 (Delay of Germination1) family plays key regulatory roles in seed dormancy and germination. However, a genome-wide analysis of DOG1 genes has not been performed for pepper (Capsicum annuum), one of the agriculturally important species, and no studies have been conducted to characterize their expression profiles. Based on C. annuum genome information, the identification and expression analysis of CaDOG1 gene family members through bioinformatics approaches can provide a theoretical foundation for subsequent studies on the biological functions of CaDOG1s and the improvement of seed traits in C. annuum breeding. In this study, a total of 13 CaDOG1 genes were identified in the C. annuum genome. Phylogenetic analysis showed that these CaDOG1s, along with DOG1s from thale cress (Arabidopsis thaliana), rice (Oryza sativa), and maize (Zea mays), were classified into four subgroups. All CaDOG1 genes were unevenly distributed on six C. annuum chromosomes, and they had relatively conserved exon–intron patterns, most with zero to one intron. According to the chromosomal distribution patterns and synteny analysis of the CaDOG1 genes, the CaDOG1 family expanded mainly through replication, which occurred predominantly after the divergence of dicotyledons and monocotyledons. Conserved motif analysis indicated that all encoded proteins contained Motif 2 and Motif 6, except for CaDOG1-3. Expression profile analysis using transcriptome data revealed that CaDOG1 genes were differentially expressed across various tissues and developmental stages, with notable involvement in flowers and seeds. Quantitative real-time PCR also revealed that all CaDOG1 genes were downregulated during seed germination, indicating that CaDOG1s may play negative roles in seed germination. Moreover, upon abscisic acid treatment, six CaDOG1 genes exhibited upregulation, while in response to ethylene, four CaDOG1 genes exhibited downregulation. Taken together, these findings provide an extensive description of the C. annuum DOG1 gene family and might facilitate further studies for elucidating their functions in seed germination.

Genetic variation analysis of EMS-induced Curcuma alismatifolia Gagnep. mutants using SSR markers

2025-06-20

Weiwei Huang , C. Tao , Qi Jiang +3 more | European Journal of Horticultural Science

Abstract Curcuma alismatifolia Gagnep. is an original ornamental flower. To induce new phenotypic variations and breed new varieties with market value, ethyl methanesulfonate (EMS) was used to treat C. alismatifolia … Abstract Curcuma alismatifolia Gagnep. is an original ornamental flower. To induce new phenotypic variations and breed new varieties with market value, ethyl methanesulfonate (EMS) was used to treat C. alismatifolia seedlings at various concentrations (0.0%, 0.1%, 0.5%, 1.0%, 1.5% and 2.0%) and for various durations (10, 15, 30, 60, 120 and 240 min). The treatment with 0.1% concentration for 10 min resulted in the highest survival rate, while treatments with 0.6% and 0.8% concentrations for 240 min could produce more diverse phenotypes. The treatment conditions of 2.0% EMS for 10 min and 0.5% EMS for 60 min are considered the semi-lethal doses for EMS mutagenesis of C. alismatifolia. This mutagenic process effectively induced diverse phenotypic changes, such as plant dwarfism, fewer bracts, leaf adhesion, chimera formation, altered coloration of the flower petal, and leaf fusion. We used a total of 16 simple sequence repeat (SSR) markers to analyze the genetic variation of the EMS-induced C. alismatifolia seedlings. The analysis revealed the following genetic parameters: the average polymorphism information content (PIC) was 0.6044, the number of alleles (Na) ranged from 2 to 3, the number of effective alleles (Ne) varied between 1.2195 and 2.9877, and the genetic diversity index (I) was 0.7532. This study promotes the mutation breeding program of C. alismatifolia. Significance of this study What is already known on this subject? Ethyl methanesulfonate (EMS) triggers mutations, while simple sequence repeat (SSR) markers are used to identify genetic variations. However, systematic research on the combined application of EMS mutagenesis and SSR analysis for breeding new varieties of Curcuma alismatifolia remains limited, particularly in optimizing treatment conditions and exploring genetic diversity in this species. What are the new findings? The study identifies the most effective EMS treatment durations for inducing diversity in C. alismatifolia : 2.0% for 10 min and 0.5% for 60 min. These treatments result in notable phenotypic changes such as dwarfism and altered leaf and flower traits. SSR analysis confirms significant genetic diversity, with an average polymorphism information content (PIC) of 0.6044 and a genetic diversity index of 0.7532 among mutants. What are the expected impacts on horticulture? The study aims to improve the breeding of C. alismatifolia by inducing genetic diversity and selecting traits such as dwarfism plants and unique leaf patterns, aiding in creating new cultivars for varied market needs.

Transcriptomic analysis reveals the drought tolerance mechanism of Larix kaempferi ERF6 (LkERF6) in Nicotiana benthamiana

2025-06-20

Ming Tian , J. G. Lu , Yuqian Wang +2 more | Plant Cell Tissue and Organ Culture (PCTOC)

Dynamic changes in DNA methylation and correlated expressing genes during somatic embryogenesis in Hevea brasiliensis

2025-06-19

Ling Li , Jin Lai Miao , Jin Liu +6 more | Plant Cell Tissue and Organ Culture (PCTOC)

Speed-bred crops for food security and sustainable agriculture

2025-06-19

Garima Aggarwal , A.S. Jeena , K. L. Mehra +7 more | Planta

Biochemical Analysis of Eight Mutants of Vicia faba L. in M2 & M3 Generation

2025-06-17

Khochava M.R. , A.D. More | International Journal of Science and Research (IJSR)

Plant epigenetics: controlling genome expression to integrate developmental and environmental cues

2025-06-17

Marco Catoni , Tamara Lechon Gomez , Aline V. Probst | PubMed

Exogenous Application of 6-Benzyladenine Increases Peanut (Arachis hypogaea L.) Pod Size by Promoting the Cell Division During the Rapid-Expansion Stage

2025-06-17

Zhenghao Lv , Pei Guo , Guohu Lan +7 more | Journal of Plant Growth Regulation

Effects of Different Concentrations of BA with NAA on Shoot Proliferation and Acclimatization of Niaw Dam Mo 37 Rice

2025-06-16

Sasiwimom Muangmee , Sompong Te- chato , Sureerat Yenchon | King Mongkut's Agricultural Journal

Niaw Dam Mo 37 rice has many important benefits, such as reducing cholesterol, inhibiting gastric acid secretion, helping blood circulation, slowing down the degeneration of body cells, and inhibiting the … Niaw Dam Mo 37 rice has many important benefits, such as reducing cholesterol, inhibiting gastric acid secretion, helping blood circulation, slowing down the degeneration of body cells, and inhibiting the growth of lung cancer cells. In the present, no authors have reported on tissue culture for the propagation and improvement of Niaw Dam Mo 37. Thus, this study aimed to investigate the germination of mature seeds, multiple shoot formation from culturing shoot tip in liquid oil palm culture medium (OPCM) with plant growth regulators and acclimatization of complete plantlets to field conditions. The results revealed that seed germination in 1 week could be divided into 3 types: shoot alone, root alone, and both shoot and root. Total germination after 7 days of cultures was 85.5 percent. For multiple shoot formation, the results showed that shoot tip culture in liquid OPCM medium supplemented with 0.5 mg/L NAA and 1.5 mg/L BA gave the highest number of shoots at 7.6 shoots, significantly different (p<0.05) from other treatments. For acclimatization, plantlets cultured on medium with 0.5 mg/L NAA had the highest survival rate at 100 percent. The investigation’s findings can be used to provide fundamental knowledge for propagation, breeding and plant genetic conservation of Niaw Dam Mo 37 rice in the future.

Occurrence, fate and impact of steroidal estrogens and xenoestrogens in plants: A review

2025-06-16

Junyue Wang , Yang Chen , Zhifei Zhang +4 more | Journal of Environmental Management

The CmCt gene induced reversibility of copper toxicity in melon

2025-06-12

Miao Huang , Chaonan Wang , Linshan Shang +5 more | Journal of Hazardous Materials

Effect of Moisture Content on Different Engineering Properties of Pigeon Pea

2025-06-12

Mahendra Daheriya , Mohan Singh , Swati Singh Maravi +3 more | Journal of Advances in Biology & Biotechnology

Pigeon pea (Cajanus cajan) is a crucial pulse crop cultivated across tropical and subtropical countries, with India being the largest producer. It plays an important role in food security, soil … Pigeon pea (Cajanus cajan) is a crucial pulse crop cultivated across tropical and subtropical countries, with India being the largest producer. It plays an important role in food security, soil enrichment through nitrogen fixation, and adaptation to marginal environments. This study characterizes the physical properties of pigeon pea grains for optimization in agricultural and processing industries. Key physical properties, including sphericity (0.82–0.90), grain weight (96–102 g), bulk density (820–890 kg/m³), actual density (1310–1340 kg/m³), coefficient of internal friction (0.4–0.7), and angle of repose (25–28°C), were studied under varied moisture conditions. The findings indicate that moisture content significantly influences these properties, impacting handling, storage, and processing efficiency. Pigeon pea’s tolerance to hostile temperatures and its agronomic features make it a favourite crop for smallholder farmers. Understanding its physical properties is essential for optimizing post-harvest processes, improving mechanization, and enhancing overall productivity in the agricultural sector.

Replication of Genomes in Eukaryotes: Analyses of the Structure and Functions of the Replicative ε Polymerases from Yeasts, Plants and Animals

2025-06-09

Peramachi Palanivelu | World Journal of Advanced Research and Reviews

In all living organisms, genome replication is an indispensable activity in their life cycle. As compared to prokaryotes, genome replication in eukaryotes is much more complex. In eukaryotes, the genome … In all living organisms, genome replication is an indispensable activity in their life cycle. As compared to prokaryotes, genome replication in eukaryotes is much more complex. In eukaryotes, the genome replication is accomplished by a more specialized, multi-protein complex known as replisome. Therefore, for a complete understanding of the replication process in eukaryotes, the structure-function relationship of their replisome complex is important. The eukaryotic replisome complex is mainly composed of 3 replicative polymerases (pols), viz. α, ε and δ. The initiation of genome replication starts with the synthesis of primers by the α pol (a primase), which is followed by the synthesis of leading- and lagging-strands by the next two replicative pols, viz. ε and δ, respectively. Among them, the pol ε is an essential enzyme that links the DNA replication machinery to S-phase checkpoint-control in eukaryotes. The active sites of pol and proofreading (PR) domains of α and δ were analyzed from yeasts, animal and plant sources and reported by this author already [1, 2]. The third replicative enzyme, viz. the pol ε is analyzed and reported here. Interestingly, the yeast, animal and plant ε pols possess almost identical pol, PR and carboxyl terminal domains (CTD) with the same active site amino acids. Their catalytic cores use the same template-binding pair (–YG-), the catalytic amino acid (K), the nucleotide selection amino acid (Q) and similar catalytic metal-binding motifs in the pol domain as reported for the other replicative pols. However, they exhibit poor identities among themselves, e.g., the human pol ε showed only 46.69% and 43.19% identities to the plant (Arabidopsis thaliana) and yeast (Saccharomyces cerevisiae) pol ε sequences, respectively. Furthermore, these eukaryotic ε pols use the same active site amino acids in the PR exonuclease domain, and belong to the DEDD(Y)-superfamily of PR exonucleases. Moreover, similar to the other two replicative enzymes, the ε pols also use similar regulatory Zn2+-binding motifs (ZBMs) in their CTD. In addition, it harbours a unique ZBM in their pol domain. Interestingly, the two invariant motifs, viz –SLYPS- and -YGDTD- which are the characteristic motifs found in the other replicative α and δ pols and B-family of DNA pols, are not found in ε pols. Many specialized, conserved sequence motifs are also identified in the ε pols and discussed.

Radiation hormesis and reactive oxygen species-mediated stress priming in plants

2025-06-06

Jin‐Hong Kim | Plant Science

Characterization of Bacillus subtilis PM49 and its Role in Drought Stress Amelioration in Linum usitatissimum L.

2025-06-02

Mah Rukh , Jehangir Khan , Nizakat Bibi +4 more | Plant and Soil

Production and use of recombinant mirnas to dissect the intrinsic differences in miRNA biogenesis and regulation of drug metabolism and disposition

2025-06-01

Meijuan Tu , Yimei Wang , Ai‐Ming Yu +1 more | Drug Metabolism and Pharmacokinetics

Significance of glutathione in the hormesis effect: a case study of the relationship between heavy metal Cd and monitoring plant Tillandsia ionantha

2025-06-01

Guiling Zheng , Jingyi Zhang , Yuanyuan Liu +2 more | Plant Physiology and Biochemistry

Molecular Characterization and Targeting Vairimorpha ceranae Rad3-like Helicase through Docking Simulations for Novel Control Strategies...

2025-06-01

Nahla A. M. Fathy , Ashraf S.A. El-Sayed , Hesham M. Abd El Halim +4 more | Bulletin of Faculty of Science Zagazig University

Qualitative and Quantitative Characterization of Mutations and Genetic Diversity Analysis in M2 Populations of Chilli (Capsicum annuum L.)

2025-06-01

Nazarul Hasan , Sana Choudhary , Neha Naaz +5 more | Crop Design

Activity of the photosynthetic apparatus and productivity of transgenic winter wheat plants with partial suppression of the proline dehydrogenase gene

2025-06-01

G.O. Priadkina , О. В. Дубровна , S.K. Sytnyk +1 more | Studia Biologica

Background. Partial suppression of the proline dehydrogenase (ProDH) gene in transgenic winter wheat plants leads to an increase in the level of free proline accumulation. However, the effect of increasing … Background. Partial suppression of the proline dehydrogenase (ProDH) gene in transgenic winter wheat plants leads to an increase in the level of free proline accumulation. However, the effect of increasing the content of this amino acid on the physiological and biochemical characteristics of this crop is still not fully understood. In this regard, the aim of the work was a comparative analysis of the influence of the free proline accumulation on the activity of the photosynthetic apparatus parameters of transgenic wheat plants at reproductive period under variable weather conditions, as well as on their productivity. Materials and Methods. The study involved non-transformed winter bread wheat plants of genotype UK 997/19 and transgenic lines of seed generation T2 obtained on their basis. The content of free proline, photosynthetic pigments and parameters of the photosynthetic apparatus activity were determined. The analysis of the elements of the crop structure was carried out at full ripeness. Results. Under conditions of increased air temperature or lack of moisture in the soil, it was established that the total chlorophyll content in the leaves of plants of the transgenic lines at milk-wax maturity exceeded its level in the wild-type plants by 15.9–32.5 %. At this phase, they had a higher effective quantum yield (ϕPSII) by 16–28 %, the coefficient of photochemical quenching (qP) by 23–26 % and the fraction of open reaction centers (qL) by 28–61 % of photosystem II (PSII). No specific regularities were found in the changes in the non-photochemical quenching parameter (NPQ) in the antenna complexes of PS II leaves of the transgenic plants relative to the wild-type ones. The grain yield of plants of the modified lines was higher than that of the wild type. A significant positive correlation was found between the grain productivity of the transgenic plants with the fraction of open reaction centers of PSII, the effective quantum yield and photochemical quenching of fluorescence parameter (the coefficient of determination of the relationship varied from 0.762 to 0.966). Conclusions. The study results indicate that the elongation of the functioning of the flag leaf during the reproductive period and the higher activity of the photosynthetic apparatus in the transgenic wheat lines with an increased proline content under the conditions of increased air temperature or lack of moisture in the soil contributed to an increase of their grain productivity.

Differential Impact of CBL3-1 and CBL3-2 Gene Silencing on Tomato Growth and Responses under Drought Stress

2025-06-01

Peyman Aghaie , Sayed Ali Hosseini Tafreshi , Amir Hossein Forghani | Russian Journal of Plant Physiology

Transcriptional changes at different developmental stages of rice (Oryza sativa L.) following lunar orbit flight

2025-06-01

Xiaohui Du , Yan Zhang , Qing Yang +2 more | Life Sciences in Space Research

Genetic Modification of Wheat to Increase Its Drought Tolerance

2025-06-01

О. В. Дубровна , S. I. Mykhalska , А. Г. Комисаренко | Cytology and Genetics

EMS-Induced Meiotic Configurations in Corchorus capsularis L.: An Arcane Phenomenon of Secondary Associations among Bivalents

2025-06-01

Girjesh Kumar , J.R. Yadav , Naveen Kumar Tiwari +2 more | Cytology and Genetics

Manipulating alternative end-joining alters carbon-ion beam-induced genome mutation profiles in Arabidopsis thaliana

2025-05-31

Jing Long , Jian Zhao , Jingmin Chen +7 more | DNA Research

Abstract DNA double-strand breaks (DSBs) repair via POLQ-mediated alternative end-joining (Alt-EJ) is error-prone and mutagenic. However, Alt-EJ is often inhibited by classical nonhomologous end-joining (C-NHEJ) or homologous recombination, the precise … Abstract DNA double-strand breaks (DSBs) repair via POLQ-mediated alternative end-joining (Alt-EJ) is error-prone and mutagenic. However, Alt-EJ is often inhibited by classical nonhomologous end-joining (C-NHEJ) or homologous recombination, the precise impact of Alt-EJ on plant genome instability remains unclear. Here, we employed carbon-ion beam (CIB) which induce complex DSBs to bias cellular repair strategies toward Alt-EJ; additionally, a specific genetic background of C-NHEJ deficiency (lig4-4) Arabidopsis thaliana line and the POLQ-deficient (teb-3 and teb-8) were combined to further amplify the mutagenic effects of CIB mediated by Alt-EJ. The lig4-4 exhibited higher sensitivity to CIB than POLQ-deficient lines. teb-8 exhibited constitutive DNA damage response (DDR), whereas DDR in lig4-4 was strictly induced by CIB. At genome scale, lig4-4 showed substantial changes in the insertion and deletion (InDels) mutation profile, with a higher proportion and larger size of InDels as well as greater microhomology dependence than wild-type. In contrast, teb-8 showed moderate changes, including increased single-base InDels and complex mutations, but lacking &gt;30 bp InDels. Loss-of-function in LIG4 and POLQ resulted in a higher proportion of high-impact genome mutations than wild-type even at lower doses. These findings offered essential insights for the development of a novel repair pathway-driven heavy ion beam mutagenesis system.

Unlocking the secret of soft X-ray impact on seed germination

2025-05-30

Sherif Hamdy , Ludivine Soubigou‐Taconnat , Audrey Dupont +4 more | Research Square (Research Square)

<title>Abstract</title> <bold>Background</bold> Seed quality analysis using X-rays is increasingly explored due to its invasive and rapid nature. Yet, the current absence of reliable and standardised imaging protocols has led to … <title>Abstract</title> <bold>Background</bold> Seed quality analysis using X-rays is increasingly explored due to its invasive and rapid nature. Yet, the current absence of reliable and standardised imaging protocols has led to contradictory effects of X-ray exposure in previous studies. Our work systematically investigated the effect of soft X-rays on a wide range of plant materials. <bold>Results</bold> The baseline of three germination categories was established across seven species before the application of soft X-ray exposure under controlled standard germination conditions. The high inter-varietal and inter-lot variabilities, in addition to the strong interaction between X-ray exposure with variety and lot, reinforced the need to consider genetic and seed quality aspects while evaluating the impacts of X-rays. A slight stimulative effect was observed on most of the species (bean, carrot, fennel, maize, radish, and ryegrass), notably, with a repeated reduction in ungerminated seeds. Intrinsic physical quality holds a crucial value where the minor negative impact observed in soybean originated from its degraded physical quality and not from X-ray exposure, hence, no destructive effects were detected. To understand whether seed size plays a significant role in a seed's response to exposure, linear regression models were built to predict 3D seed traits (volume) from 2D X-ray images. Yet, seed size did not explain the variation in responses to soft X-rays. However, the average density of the seven species explained both their natural germination (<italic>p</italic> < 0.01; R²=0.82) and their germination outcomes after exposure (<italic>p</italic> < 0.01; R²=0.88). Among all species, fennel with notably low density (0.7 g/cm³) demonstrated the most pronounced gains in germination after exposure (4.6 ± 6.3%) due to the stimulative effect. <bold>Conclusion</bold> Soft X-ray exposure is non-destructive with a beneficial effect on germination but can be strongly influenced by underlying genetics and the physical quality of the tested seeds. This study adopted internationally-standardised germination procedures and tested the effect of soft X-rays across diverse botanical, genetic and seed quality profiles. This work addressed important gaps in evaluating X-ray impacts and proposed a robust design and well-examined radiography protocol for a proven non-destructive seed quality analysis.

Molecular characterization of T-, C- and S- specific cytoplasmic male sterile (CMS) maize inbreds using mitochondrial SSRs for its utilization in baby corn breeding

2025-05-30

Saikat Pal , Rajkumar U. Zunjare , Vignesh Muthusamy +7 more | Molecular Biology Reports

Fine-Tuning EMS Treatments to Produce Large Sorghum Mutant Populations for FIND-IT

2025-05-29

Patrick J. Mason , Anko Blaakmeer , Agnelo Furtado +7 more | Research Square (Research Square)

<title>Abstract</title> Chemical, biological, and physical mutagens induce modifications of nucleotides in the exposed organisms, resulting in base changes in their DNA. When harnessed, mutagenesis increases genetic diversity in crops and … <title>Abstract</title> Chemical, biological, and physical mutagens induce modifications of nucleotides in the exposed organisms, resulting in base changes in their DNA. When harnessed, mutagenesis increases genetic diversity in crops and aids in elucidating gene function through the study of mutants with altered phenotypes, particularly when combined with reverse genetic techniques such as Fast Identification of Nucleotide variants by droplet digital PCR (FIND-IT). Sorghum (<italic>Sorghum bicolor</italic>) is a prime candidate organism for mutagenesis; several mutant populations have been produced using ethyl methanesulfonate (EMS) as the mutagen. However, none of these studies address the optimisation of EMS treatment rates for large sorghum mutant populations. Here, we examined how varying EMS concentrations (0.05, 0.15, and 0.25%) plus a zero control affect agronomic traits, including plant survivability, seed yield, pest burden, and growth rates, as well as the number of induced genomic variants in the inbred sorghum variety BTx623. Whole-genome sequencing and variant analysis of M3 plants showed a positive correlation between EMS concentration and mutation load, although significant gains diminished beyond 0.15%. An EMS rate of 0.25% led to very poor seed yields, whilst 0.05% generated too few variants to achieve genome mutant saturation. Regression analyses indicate that a 0.10% EMS treatment may best balance seed yield and mutation load, and enable construction of large-scale mutant libraries for FIND-IT. This work underscores the importance of optimizing EMS concentration when producing sorghum mutant populations and provides insights into the effects of EMS on plant agronomic performance, germination, and mutation profiles.

In Silico Identification and Verification of the Anticancer Mechanism of TMBM-010 from Oxytropis herba with a Rational Delivery System Design

2025-05-29

Zhe Zhang , Huijuan Zheng , Jindian Fan +7 more | ACS Applied Bio Materials

Patients diagnosed with gastric cancer often face poor prognoses and limited treatment options. Current therapies remain limited, resulting in significant adverse effects and suboptimal outcomes. Network pharmacology analysis suggests that … Patients diagnosed with gastric cancer often face poor prognoses and limited treatment options. Current therapies remain limited, resulting in significant adverse effects and suboptimal outcomes. Network pharmacology analysis suggests that TMBM-010, a natural compound, holds the potential to modulate key pathways in cancer progression. Through network pharmacological analysis, we identified the anticancer mechanisms of TMBM-010, including ROS induction, DNA damage, apoptosis, and inhibition of DNA repair pathways. To enhance the bioavailability and efficacy of TMBM-010, we developed TMBM-010-loaded nanoparticles (TNPs) and biomimetic nanoparticles (TNPs@RGD-CM) coated with gastric cancer cell membranes and RGD ligands. TNPs@RGD-CM demonstrated high stability, excellent biosafety, and a controlled release profile. In a gastric cancer xenograft model, TNPs@RGD-CM significantly improved the bioavailability, increased ROS generation, and enhanced anticancer effects. Our findings demonstrate that TNPs@RGD-CM augment TMBM-010's bioactivity in vivo, effectively targeting cancer cells and suppressing tumor-promoting pathways. These results suggest that TNPs@RGD-CM represent a promising nanomedicine strategy for gastric cancer treatment.

Research on abnormal spermatogenesis in mouse model of Down syndrome

2025-05-29

T Wakayama , Chompusri Nattapran , T. Sugawara +3 more | Journal of Reproductive Immunology

Keragaman Genetik Aksesi Tanaman Kunyit (Curcuma longa L.) Berdasarkan Penanda Molekuler Simple Sequence Repeat (SSR)

2025-05-28

Muthia Hanin Afidani , Noor Farid , Eka Oktaviani

A plant breeding program for turmeric (Curcuma longa L.) development is required to fulfil the pharmaceutical industry's demand. Understanding genetic diversity is the initial step in selecting the parents in … A plant breeding program for turmeric (Curcuma longa L.) development is required to fulfil the pharmaceutical industry's demand. Understanding genetic diversity is the initial step in selecting the parents in plant breeding. Selection assisted by molecular markers, specifically Simple Sequence Repeat (SSR), can be used to obtain genetic information. However, genetic information on turmeric based on SSR markers in Indonesia is still limited. This study aimed to determine the genetic diversity and relationship of six accessions of turmeric using 4 (four) SSR primers. This study was conducted from October 2023 to January 2024 at the Laboratory of Plant Breeding and Biotechnology, Faculty of Agriculture, Universitas Jenderal Soedirman. The result showed that the CuMiSat-19, CuMiSat-20, and CuMiSat-29 primers were informative based on the electrophoresis band polymorphism. The CuMiSat-19 primer generated the highest PIC value (0,88), followed by CuMiSat-20 (0,80) and CuMiSat-29 (0,74). While CuMiSat-23 did not produce polymorphism, the PIC value was 0. The dendrogram classified six turmeric accessions into the two clusters with a Jaccard correlation coefficient range of 0.11-0.33. Cluster I consisted of accession A (Ponorogo), accession C (Gresik), accession D (Indramayu), and accession B (Semarang). Cluster II grouped accession E (Banyumas) and accession F (Bogor). Based on the polymorphism and similarity coefficient, turmeric accessions have a wide range of genetic diversity. Turmeric in different clusters is a potential germplasm that can be utilized to broaden genetic diversity in plant breeding programs.

HAMILTONIAN CYCLES IN WIJAYA KUSUMA FLOWER GRAPH

2025-05-25

Susilawati Nurdin , Dinda Khairani Nasution

In 1856, William Rowan Hamilton introduced the Icosian game. From this game, the concept of a Hamiltonian graph is defined. Hamiltonian graph is a graph that contains the Hamiltonian cycle, … In 1856, William Rowan Hamilton introduced the Icosian game. From this game, the concept of a Hamiltonian graph is defined. Hamiltonian graph is a graph that contains the Hamiltonian cycle, which is a cycle that passes through each vertex exactly once. We constructed a new class of graph which is inspired by the Wijaya Kusuma flower. In this article, we study the Hamiltonian properties of the Wijaya Kusuma flower graph. Based on the proof, it is concluded that the Wijaya Kusuma flower graph is a Hamiltonian graph.

Cytogenetic changes in Rosa spinosissima L. and Leymus angustus (Trin:) Pilg. growing under radioactive contamination conditions at the Semipalatinsk nuclear test site

2025-05-22

K. S. Minkenova , Arailym Serik , A. V. Panitskiy

In this study, we investigated the cytogenetic effects of exposure to chronic radioactive contamination on the populations of Rosa spinosissima L. and Leymus angustus (Trin.) Pilg. growing in fields affected … In this study, we investigated the cytogenetic effects of exposure to chronic radioactive contamination on the populations of Rosa spinosissima L. and Leymus angustus (Trin.) Pilg. growing in fields affected by radioactive water streams from the ‘Degelen’ test location of the Semipalatinsk test site. The results revealed that the radiation dose absorbed by these plants varied from 108 to 1,150 µGy/day, depending on the sampling points of the plants. The main exposure dose received by the plants was from 90 Sr and 137 Cs. In both plant species, chromosomal aberrations were the main contributors to the range of cytogenetic effects (double bridges and double fragments). The proportions of chromosomal aberrations were the highest among all cytogenetic effects at 42 and 54% in R. spinosissima and L. angustus , respectively. A linear relationship was established between the increase in the frequency of aberrant cells and the increase in the rate of radiation dose absorption in R. spinosissima for the entire range of the absorbed doses in question up to 1,129 µGy/day and in L. angustus for the range of absorbed doses from 152–583 µGy/day.