Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism

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No publication fee; no access fee. Plant Science Latest Protocol Video. Author Map. Protocols in Current Issue. By Date By View. Protein stability can be determined after treatment with the protein synthesis inhibitor Cycloheximide. Cycloheximide is a translational inhibitor that inhibits protein synthesis via cytoplasmic ribosomes.

Here we This protocol provides an efficient method to purify laccases from rice stems. His research program heavily utilizes plant natural variation and integrates diverse fields such as genomics, metabolomics, biochemistry, computational biology and molecular evolution.

June Nasrallah Professor. June Nasrallah obtained her B. Kevin Nixon Professor. Kevin Nixon has diverse research interests in the theory and practice of plant systematics. His taxonomic interests include higher level analysis of seed plant and angiosperm relationships, and relationships of Hamamamelid and Rosid ordinal and family relationships. Kevin works at the generic and species level within Fagaceae, and in particular in Quercus. Wojtek Pawlowski Associate Professor. Miguel Pineros Adjunct Associate Professor.

Roots are the essential organ for plant nutrition, absorbing water and nutrients. Research in the Pineros lab focuses on the role of two distinct, but complementary aspects of root biology and plant adaptation to environmental stresses: root system architecture and membrane transport. Applying a combination of approaches such as electrophysiology, molecular biology, cellular, and whole plant physiology, we are elucidating plant responses mediating calcium signaling, crop acid soil resistance, and mineral transport.

Adrienne Roeder Associate Professor. Adrienne Roeder is fascinated by how beautiful and complex patterns form during development. The patterning process generally requires that one cell adopts a different identity from its neighbor. Patterns are generally formed while the cells are growing and dividing, yet the coordination of cell division and growth with the process of patterning is only beginning to be understood.

Researchers in the Rose lab investigate the formation, function, and evolution of plant structural polymers, as well as extracellular processes associated with developmental and environmental responses. We use a broad range of analytical approaches, including genomic and proteomic technologies, working with experimental systems that range from tomato fruit to algae, to study cell wall and cuticle biology.

At the spatial level we are interested in cell and tissue type specialization, particularly at the surface of plant organs. Michael Scanlon Professor. Research in the Scanlon lab focuses on mechanisms of plant development and evolution of plant morphology. Utilizing comparative developmental genetics and functional genomics, he is especially interested in the processes whereby meristems make leaves and embryos make meristems. Thomas Silva Senior Lecturer. Tom Silva's primary focus is in the instruction of plant biology courses for majors and non-majors.

Chelsea Specht Professor.

The Specht lab uses morphological and developmental techniques combined with molecular genetics, comparative genomics, and evolutionary biology to study the natural diversity of plants and better understand the forces creating and sustaining this diversity.

This research incorporates elements of systematics, developmental genetics and molecular evolution to study the patterns and processes associated with plant speciation and diversification. David Stern Adjunct Professor. The underlying research themes in the Stern laboratory are chloroplast biology, bioenergy and nuclear-cytoplasmic interactions.

Within this framework, they study how chloroplast genes and metabolic activities are regulated by the products of nuclear genes, usually acting at the transcriptional or post-transcriptional level. Areas of emphasis include the roles of ribonucleases and RNA-binding proteins and assembly of the carbon-fixing enzyme Rubisco. Dennis Stevenson Adjunct Professor. Dennis Stevenson's major research interests in the past few years have focused upon the evolution and classification of the Cycadales cycads and their placement in seed plant phylogeny.

Previously, DNA sequencing was performed almost exclusively by the Sanger method, which has excellent accuracy and reasonable read length but very low throughput. Sanger sequencing was used to obtain the first sequence of the human genome in Lander et al. Shortly thereafter, the second complete individual genome James D. Watson was sequenced using next-generation technology, which marked the first human genome sequenced with new Next Generation Sequencing NGS technology Wheeler et al. Therefore, NGS reads DNA templates in a highly parallel manner to generate massive amounts of sequencing data but, as mentioned above, the read length for each DNA template is relatively short 35— bp compared to traditional Sanger sequencing — bp.

NGS technologies have increased the speed and throughput capacities of DNA sequencing and, as a result, dramatically reduced overall sequencing costs Metzker Genome-based sequencing yielding genomic deletions and rearrangements, copy-number variations CNV of smaller regions or elements, and single-nucleotide polymorphisms SNPs. RNA-Sequencing, yielding genome-wide and quantitative information about transcribed regions exons, and subsequently transcripts.

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Chromatin-immunoprecipitation ChIP -Seq. The inclusion of NGS-based transcriptome sequencing for ChIP of transcription factor binding and epigenetic analyses usually based on DNA methylation or histone modification ChIP completes the picture with unprecedented resolution enabling the detection of even subtle differences such as alternative splicing of individual exons.

Next-generation sequencing technologies have found broad applicability in functional genomics research. Their applications in the field have included gene expression profiling, genome annotation, small non-coding RNA ncRNA discovery and profiling, and detection of aberrant transcription, which are areas that have been previously dominated by microarrays. Thus, functional genomics and systems biology approaches will benefit from the enormous data density intrinsic to NGS applications, which will beyond doubt play an important role both in definition as well as verification of mathematical models of biological systems such as a cell or a tissue.

As mentioned above the inventory of methods used to study gene product functions in vivo i. However, there are some limitations inherent to this type of approach. First of all, physiologically essential genes cannot be switched off, and the induced mutagenesis can lead to concomitant mutations. The use of microarrays can lead to misinterpretation of the results since changes in transcription are not always accompanied by changes in protein level Mittler et al. Moreover, the transcription level fails to reflect post-translation modifications of protein products which often occur in vivo.

It is also worth to mention that when an enzyme possesses many isoforms, it is difficult to measure the activity of each of them in vivo Slakeski et al. In view of the above-mentioned limitations, development of novel models for functional genetics which will aid to overcome these difficulties is deemed very much desirable.

One of such models may be the approach that is developed in our laboratories that employ transgenic plants that constitutively express bacterial genes, which code enzymes that are functionally homologous to plant enzymes. Such an approach was proposed and used in our laboratory since mids Piruzian et al. It involves several stages: search a cloning of a gene of interest, sequencing, sequence modification if needed, e. Such an approach is feasible owing to the similarity of metabolic pathways and gene networks that regulate the activities of pro- and eukaryotic organisms under normal conditions and under exposure to various biotic and abiotic stresses.

Activation tagging has been used for the isolation of mutants with resistance to biotic stress. For example, CDR1-D is a mutant that is resistant to sprayed suspensions of virulent Pseudomonas syringae pathovar tomato Pst Xia et al. CDR1 encodes an extracellular aspartic protease, which is a member of a large family of aspartic proteases in Arabidopsis. CDR1 functions in the production of a systemic signal that induces basal defenses.

This phenotype is the result of the overexpression of a gene that encodes a class 3 FMO protein. Recently we have proposed the model for studying the role of plant dioxygenases. Phenolic compounds serve as antioxidants and protect plants from active oxygen species. The content of phenolic compounds changes as plants grow and get mature and in response to biotic and abiotic influences, and these changes are achieved through modulation of enzymatic activities involved in their synthesis and degradation.

Enzymes that take part in oxidation of aromatic compounds include dioxygenases Tsoi et al. These enzymes oxidize phenolic compounds by breaking the aromatic ring, and thus enable subsequent biodegradation of phenols. There is evidence that plant dioxygenase coded for by the lls gene of maize may participate in the hypersensitive response of the plant to a pathogen attack Lawton and Maleck For a study of the role played by dioxygenase in plants we have chosen the bacterial gene nah C Y of Ps. Our choice was due to the fact that this enzyme possesses broad substrate specificity and can also use pyrocatechin as substrate Tsoi et al.

The expression of bacterial 1,2-dehydronaphthalene dioxygenase coded by the nah C gene in tobacco plants resulted in marked phenotypic and morphologic changes: chlorosis of the leaves, development of necrotic spots, delayed rooting and growth, and early flowering Piruzian et al. Data on expression of bacterial 1,2-dihydroxynaphtalene dioxygenases in plants have not been reported in the literature.

The necrotic spots on leaves of transgenic plants could have resulted from accumulation of phenolic substances. The above-mentioned phenotype and morphology changes suggested that the expression of bacterial dioxygenase resulted in alteration of the level of phenolic compounds in the transgenic plant cells.

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Measurements of phenolic acid content indicate that normal metabolism of phenolic compounds is disturbed in the plants, and the disturbance apparently results in induction of a stress response and appearance of the necrotic spots. In our opinion, such transgenic plants are a promising model for the study of mechanism of genome functioning under normal conditions and under stress, as well for the study of functions of phenolic compounds.

Environmental stresses are the major factors adversely affecting plant growth and development as well as productivity. Of the various abiotic stresses, drought and osmotic stress cause considerable agronomic problems by limiting crop yield and distribution world-wide Chaves and Oliveira Drought and osmotic stress induce a range of alterations at the molecular, biochemical, and cellular levels in plants, including stomatal closure, repression of photosynthesis, accumulation of osmolytes, and the inducible expression of genes involved in stress tolerance Shinozaki and Yamaguchi-Shinozaki The accumulation of proline by plants is a common physiological indicator and occurs under various abiotic stresses.

There is an increasing body of evidence supporting the role of proline as a compatible osmolyte that maintains cellular osmotic adjustment and stabilizes the structure of proteins and membrane integrity Verbruggen and Hermans Overexpression of different genes has been shown to significantly enhance proline levels in transgenic rice and improve their tolerance to environmental stresses Ito et al.

Transgenic Plants as a Tool for Plant Functional Genomics

The transference of a single gene encoding a specific stress protein does not always result in sufficient expression to produce useful tolerance, because multiple and complex pathways are involved in controlling plant drought responses Bohnert et al. Targeting multiple steps in a pathway may often modify metabolite fluxes in a more predictable manner. Another promising approach is therefore to engineer the overexpression of genes encoding stress inducible transcription factors.

There is increasingly more experimental support for the manipulation of the expression of stress-related transcription factor genes as a powerful tool in the engineering of stress-tolerant transgenic crops. This would, in turn, lead to the up-regulation of a series of stress-related genes under their control in transgenic plants P. Agarwal et al. Following the application of microarray technology, several hundred stress induced genes, mainly in the model plant Arabidopsis thaliana , have been identified as candidates for manipulation Shinozaki and Yamaguchi-Shinozaki and have been classified into three groups Bhatnagar-Mathur et al.

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Examples include enzymes for synthesis of osmoprotective compounds, late embryogenesis abundant LEA proteins, osmotins, chaperons, channels involved in water movements through cell membranes, ubiquitins, proteases involved in protein turnover, and detoxifying enzymes; b genes with as yet unknown functions; and c regulatory genes, such as those coding for kinases, phosphatases and transcription factors. Mutants with abiotic stress tolerance have been isolated by activation tagging and include the edt1 mutant recently identified under drought conditions Ahad et al.

This mutant showed a drought tolerant phenotype and reduced stomatal density. The enhanced drought tolerance of edt1 was associated with an increase in the expression of the gene that encodes the transcription factor HDG The overexpression of ArabidopsisHDG11 in tobacco can also confer drought tolerance and reduced leaf stomatal density Zhang FOX lines that consist of 43 stress-inducible transcription factors were constructed to elucidate stress-related gene function Fujita et al. The T1 generation was screened for salt-stress-resistant lines and led to the identification of salt-tolerant lines.

The overexpression of AtZIP60 leads to the upregulation of stress related genes, which suggests an important role for this transcription factor in stress-responsive signal transduction. Transcription factors play an important role in plant development and stress responses. The Arabidopsis genome encodes more than 1, transcription factors. Zhang Weiste and colleagues Weiste et al. They constructed a destination vector to enable ectopic expression driven by the CaMV35S promoter and included a HA-tag sequence to reveal transgene-specific expression. This approach yielded eight plants that show enhanced tolerance to oxidative stress resulting from the overexpression of the same ERF.

Typically a gene coding for a transcription factor in Arabidopsis is isolated, characterized and shown to improve drought response when overexpressed. The gene is then transferred to a crop plant where it often confers the same drought-tolerant phenotype.

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Arabidopsis plants with a gain-of-function mutation in the HRD gene hrd-D mutants are drought resistant, salt-tolerant, and overexpress abiotic stress marker genes. These plants also show enhanced photosynthetic assimilation and reduced transpiration Pereira et al. HRD gene overexpression conserves drought tolerance in both dicots and monocots. In other cases a gene coding for a transcription factor is isolated and characterized in Arabidopsis , but its orthologue gene in the crop plant of interest is identified and made to overexpress. For example Nelson et al.

Nelson et al. A high-throughput gain-of-function approach has been applied to isolate salt stress tolerance genes using cDNAs of Thellungiella halophila Du et al. Thellungiella halophila is a type of salt cress similar to Arabidopsis that can grow under high salt conditions. The cDNA library was prepared after salt stress treatment.

Novel salt stress tolerance genes were isolated from this mutant collection. Ethylene response factor ERF genes have been successfully introduced into rice, generating transgenic rice with enhanced tolerance to biotic and abiotic stresses. Overexpression of transcription factor Sub1A-1 in a submergence-intolerant Oryza sativa ssp. By overexpressing a Athsp protein, Katiyar- Agarwal and associates generated a heat-tolerant transgenic rice cv.

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Pusa basmati 1 line. This group showed that almost all the transgenic plants recovered after severe heat stress of 45—50 0 C and exhibited vigorous growth during the subsequent recovery at 28 0 C, while the untransformed plants could not recover to a similar extent. In our experiments with salt stress tolerance, we have selected a mutant of E.

The mutation, which consists of single amino acid substitution leucine is replaced by glycine caused a conformational change in the regulatory region of the protein, made the enzyme less sensitive to the feedback inhibition by proline. The rationale for using a mutant form of the prokaryotic proteins which determine a strictly define phenotype, osmotolerance, was that the phenotype of the plant model will be easy to assay. The expression of the osmotolerance phenotype in the model eukaryotic organism would be indicative that the prokaryotic protein is an ortholog of the eukaryotic protein.

Thus, our results demonstrate usefulness of the proposed model and the possibility of simulating the activity of a bi-functional plant enzyme with two bacterial enzymes. In Arabidopsis , knockout or silencing of HSP caused loss of the acquired thermotolerance, whereas the overexpression of HSP in transgenic plants improved tolerance to high temperature stress Gurley ; Hong and Vierling Agarwal and co-workers provided evidence that AtHSP and OsHSP impart thermoprotection to yeast cells by dissolution of heat-induced protein aggregates.

High-temperature-tolerant rice plants have also been produced by overexpressing a rice small heat-shock protein sHSP Oxidative stress may accompany heat stress by the formation of ROS Foolad et al. More recently, Qi and associates have reported that mtHsp70 over-expression suppresses programmed cell death PCD by maintaining mitochondrial membrane potential and preventing ROS signal amplification in rice protoplasts.

Koh and co-workers reported that knockout KO mutants of rice OsGSK1 , an orthologue of Arabidopsis BIN2, showed enhanced tolerance to several abiotic stresses including high temperature. Feng and associates raised transgenic rice plants overexpressing rice sedoheptulose-1,7-bisphosphatase SBPas e. They showed that overexpression of SBPase resulted in enhanced tolerance of growth and photosynthesis to high temperatures in transgenic rice plants.

Compared to wild type tobacco, the transgenic seedlings showed higher tolerance to temperature stress. Major efforts have been made to identify genes that are associated with drought stress in a number of plant species Gong et al. As a result, hundreds of genes that were induced or suppressed by drought stress have been identified.

Identification of lipolytic enzymes involved in remodeling of glycolipids

A number of these genes have been analyzed in detail, resulting in their characters as regulatory genes, such as transcription factor TF and protein kinase encoding genes, whose products regulate other stress-responsive genes. Some of the identified stress-responsive genes are functional genes which encode metabolic components, such as late embryogenesis abundant LEA proteins and osmoprotectant-synthesizing enzymes, important for stress tolerance Yang et al. Recently, Yang and associates classified drought-responsive genes into three groups based on their biological functions: transcriptional regulation, post-transcriptional RNA or protein phosphorylation, and osmoprotectant metabolism or molecular chaperons.

Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism
Plant Molecular Biology: Molecular Genetic Analysis of Plant Development and Metabolism

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