Carol Potenza

Targeting transgene expression in research, agricultural, and environmental applications: Promoters used in plant transformation

SummaryPlant genetic engineering has contributed substantially to the understanding of gene regulation and plant development, in the generation of transgenic organisms for widespread usage in agriculture, and has increased the potential uses of crops for industrial and pharmaceutical purposes. As the application of geneticallly engineered plants has widened, so has the need to develop methods to fine-tune control of transgene expression. The availability of a broad spectrum of promoters that differ in their ability to regulate the temporal and spatial expression patterns of the transgene can dramatically increase the successful application of transgenic technology. Indeed, a variety of promoters in necessary at all levels of genetic engineering in plants, from basic research discoveries, concepts and question to development of economically viable crops and plant commodities, to addressing legitimate concerns raised about the safety and containment of transgenic plants in the environment. This review covers the characterization and usage of a broad range of promoters employed in plant genetic engineering, including the widespread use of plant promoters with viral and plant origin that drive constitutive expression. Also covered are selected tissue-specific promoters from fruit, seed and grain, tubers, flowers, pistils, anther and pollen, roots and root nodules, and leaves and green tissue. Topics also include organellar promoters, and those found in specific cell types, as well as the development and evaluation of inducible (endogenous and exogenous origin) and synthetic plant promoter systems. Discussions on the relevance and potential pitfalls within specific applications are included.

Genes induced during early response to Meloidogyne incognita in roots of resistant and susceptible alfalfa cultivars

A cDNA library made to RNA from roots of Meloidogyne incognita (root-knot nematode) susceptible alfalfa cv. Lahontan seedlings 72 h after root-knot nematode inoculation was differentially screened with cDNA made from uninoculated control and M. incognita infested (72 h) root RNA. Of the six cDNAs isolated, the deduced amino acid sequences of four showed significant homology to sequences present in the databank, while two were pioneer sequences. The four cDNAs with matches to known sequences include those for glycine-rich protein, the gluconeogenic pathway enzyme phosphoenolpyruvate carboxykinase, an isoflavone reductase-like protein, and metallothionein. We have followed the expression of these genes during the course of nematode infection in both the susceptible and resistant host and also in different plant organs. Based on these analyses, the genes induced early in nematode infection are related either to metabolic pathways or to stress/defense.

Resistance gene analogue markers are mapped to homeologous chromosomes in cultivated tetraploid cotton

Degenerate primers designed from conserved motifs of known plant resistance gene products were used to amplify genomic DNA sequences from the root-knot nematode (Meloidogyne incognita) resistance genetic source, Upland cotton (Gossypium hirsutum) cultivar Auburn 634 RNR. A total of 165 clones were isolated, and sequence analysis revealed 57 of the clones to be novel nucleotide sequences, many containing the resistance (R)-protein nucleotide-binding site motif. A cluster analysis was performed with resistance gene analogue (RGA) nucleotide sequences isolated in this study, in addition to 99 cotton RGA nucleotide sequences already deposited in GenBank, to generate a phylogenetic tree of cotton R genes. The cotton RGA nucleotide sequences were arranged into 11 groups and 56 sub-groups, based on genetic distances. Multiple sequence alignments were performed on the RGA sequences of each sub-group, and either the consensus sequences or individual RGA sequences were used to design 61 RGA-sequence-tagged site primers. A recombinant inbred line (RIL) population of cultivated tetraploid cotton was genotyped using RGA-specific primers that amplified polymorphic fragments between the two RIL parents. Nine RGA markers were mapped to homeologous chromosomes 12 and 26, based on linkage to existing markers that are located on these chromosomes.

A TRANSGENE FOR HIGH METHIONINE PROTEIN IS POSTTRANSCRIPTIONALLY REGULATED BY METHIONINE

Summaryβ-Zein is one of the seed storage proteins of maize that is high in methionine (Met). In alfalfa, the β-zein gene driven by the CaMV 35S promoter showed an 8-fold lower level of transcript and protein when compared with the level in tobacco transformed with the same gene construct. The reporter gene (GUS) driven by the CaMV 35S promoter showed only a 4-fold difference between alfalfa and tobacco, suggesting that the expression of the β-zein gene is posttranscriptionally regulated in alfalfa. Callus of alfalfa transformats with the β-zein gene construct treated with exogenous Met, showed a significant increase in the β-zein level, suggesting that free Met may be limiting in the synthesis of β-zein in alfalfa. The introduction of the Arabidopsis thaliana cystathionine γ-synthase (AtCγS) gene driven by the CaMV 35S promoter into alfalfa showed a significant increase in the level of free Met and its metabolite, S-methyl methionine (SMM), but not in the bound fraction. Coexpression of AtCγS and β-zein in alfalfa increased the level of β-zein transcript and protein and decreased free Met, which suggests that the β-zein is posttranscriptionally regulated by free Met. The expression of AtCγS in tobacco did not produce a significant increase in free Met or SMM and coexpression of AtCγS and β-zein did not result in changes in the β-zein level. The results demonstrate the efficacy of the synergistic approach of increasing both the sink and the source for increasing the levels of high Met β-zein.

Plants, genes, and crop biotechnology

It seems to be both coincidence and fate that a request to review Maarten J. Chrispeels and David E. Sadava’s second edition of Plants, Genes, and Crop Biotechnology should coincide with the World Summit on Sustainable Development (WSSD) in Johannesburg, South Africa. But fate is terrible for the people of droughtand famine-ravaged nations in the same region, as their governments reject aid from the United States because genetically enhanced products have been included in the mix of grains and food. Once again, ignorance, politics and the loudest, most strident anti-biotech voices have won a short-term and shallow ‘victory’ in what can easily be described as a war on genetically enhanced organisms (GEOs). Unfortunately, these anti-biotech groups use over-the-top melodrama to garner attention and coverage, while many scientists—who do not have the time, resources, contacts, and, yes, the authority—wonder what they can do to educate the populace. While many rational individuals realize that just because someone is loud, does not mean they are right, they may still be confused because they do not understand the science of new crop technologies. Chrispeels and Sadava’s textbook is a good place for educators to start to teach people who are already affected by GEOs (or who will be in the near future), about the science behind the rhetoric, one classroom at a time. For anyone seriously contemplating the use of this book in class, the easiest way to get a good overview is to look over the contents and chapter headings, which are nicely detailed. The book also has some clearly grouped chapters that can be taught as sections. The first five chapters almost stand alone in themselves. In fact, every student starting at age 14 (High School, USA) should be required to read and understand this portion of the book. These chapters deal with population and food, regionally and globally, with emphasis on food production, sustainability, and food security. Up-to-date numbers are presented graphically, in tables, and illustrated, sometimes devastatingly, in pictures of hungry people or overused land. These numbers do not just represent hectares of millet grown or kilograms of corn produced, but vary widely in topic, including reproduction rates in developed and developing countries, per capita calorie availability, trends in aid funding, and the occurrence of anemia in preschool children and pregnant women. Two very interesting topics include the prevalence of women in agriculture and cultural prejudices that keep them food-insecure, and how the change in global political climate with the end of the Cold War has changed the pattern of aid monies given to developing nations. Each chapter is concluded with a list of discussion questions that deal with issues at local and global levels. In fact, one of the most refreshing aspects of the book is the fact that it was not written with a strong Euroor US-centric view. Instead, questions start with ‘What would be the best way for your country. . .?’ There is some repetition within these five chapters, which usually involves going into more depth on a revisited topic; but repetition can be good, especially for students in developed nations who do not have to deal with food insecurity. As a geographically challenged American, I also would have liked an accompanying world map to readily refer to, because many of the examples given throughout the book take you to exotic geographic locations to make their point. Chapters 6–10 can also be grouped together as they deal with the molecular biology and biochemistry of plants and their contribution to human/animal nutrition. This section is critical for a basic understanding of agriculture and biotechnology. However, this section of the book becomes more difficult to prescribe to nonscience majors and does not succeed as well as the first five chapters because it appears to review the molecular biology and biochemistry it covers, rather than to introduce it. For example, Chapter 6, ‘The Molecular Basis of Genetic Modification and Improvement in Crops’ is a fly-though of DNA, proteins, genes and structure, inheritance at a genetic level, recombinant DNA technology, and plant transformation, contrasting detailed molecular structure of nucleotides with flat, two-dimensional circles that represent cloning. Many of the figure sources are taken from an excellent genetics text (Hartl and Jones, also published by Jones and Bartlett), but appear to be put together hurriedly, and consequently may confuse a student with no background in these subjects. Chemical structures, electron micrographs, and schematic biochemical pathways may frighten the scientific novice more than educate him or her. Because of this, it could be recommended that someone with a strong background in plant molecular biology and biochemistry should be tapped as an instructor for this section of the text. One other recommendation that may be useful in future editions: Chapter 7 (‘Plants in Human Nutrition and Animal Feeds’) ends with a list of complementary websites. Much of the molecular and biochemical information presented in this section is also available in excellent scientific websites. Inclusion of some of the most relevant websites at the end of each chapter might be beneficial. Chapters 11 and 12 shift gears and send us underground, taking us from soil profiles to molecular soil ecology and the interaction of plants and organisms in the rhizosphere. Reading this as a novice myself, I found that these chapters are written in a cohesive and flowing manner, with a bit of humor, even though the presentation of information as more of a review than an introduction is still prevalent. Chapter 13, ‘Ten Thousand Years of Crop Evolution’ was one of my favorites and one that could be easily grouped with the first five chapters of this text as ‘need to know’ information for all students in any major. The author takes a great deal of archeological, molecular, and genetic information, and presents a neat picture of crop domestication. The truly special aspects of this chapter are the figure legends and wonderful examples showing how recognizable crops came about in human cultures. This includes contrasting the growth habits of maize and its probable wild progenitor, teosinte, to explaining that the forced hybridization of plants can lead to new In Vitro Cell. Dev. Biol.—Plant 39:56–57, January–February 2003 DOI: 10.1079/IVP2002374 q 2003 Society for In Vitro Biology 1054-5476/03

Plant Biotechnology – The Genetic Manipulation of Plants

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Induction of Genes Encoding Phenylpropanoid Biosynthetic Enzymes in Soybean Roots Inoculated with B. japonicum, Requires Nod Gene Induction and Occurs Independent of Any Known Host Functions

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The 3′ untranslated region of a soybean cytosolic glutamine synthetase (GS1) affects transcript stability and protein accumulation in transgenic alfalfa

To address the possible roles of phenylpropanoid compounds in events occurring in roots following inoculation with the compatible symbiont, we have monitored expression of the gene members encoding phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI) during nodule development in soybeans. Plant and bacterial mutants that arrest nodule development at defined stages were analyzed to correlate changes in expression of PAL, CHS and CHI genes with distinct events in nodule development. Our results suggest that induction of the ‘symbiosis specific’ PAL and CHS gene members occurs prior to any known host responses like root hair curling, infection thread formation and cortical cell proliferation. Furthermore, our results show a direct correlation in the level of PAL and CHS transcripts with the number of successful nodule foci suggesting that the resulting phenylpropanoid compounds may play a role in cortical cell proliferation associated with nodule development.