Atrium plants: proper care brings the outdoors in.
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Architecture↗
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Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a 'shooter mutant' A. tumefaciens.
In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a 'shooter' mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of 'shooter' strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4-5.7 shoots per explant). With pre-selection for the ipt (+) phenotype the transformation frequency was 24-53%, while without selection 12-28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.
Agrobacterium tumefaciens↗
Development of PPO inhibitor-resistant cultures and crops.
Recent progress in the development of protoporphyrinogen oxidase (PPO, Protox) inhibitor-resistant plant cell cultures and crops is reviewed, with emphasis on the molecular and cellular aspects of this topic. PPO herbicide-resistant maize plants have been reported, along with the isolation of plant PPO genes and the isolation of herbicide-resistant mutants. At the same time, PPO inhibitor-resistant rice plants have been developed by expression of the Bacillus subtilis PPO gene via targeting the gene into either chloroplast or cytoplasm. Other attempts to develop PPO herbicide-resistant plants include conventional tissue culture methods, expression of modified co-factors of the protoporphyrin IX binding subunit proteins, over-expression of wild-type plant PPO gene, and engineering of P-450 monooxygenases to degrade the PPO inhibitor.
Crops, Agricultural↗
Hsp90 as a capacitor of phenotypic variation.
Heat-shock protein 90 (Hsp90) chaperones the maturation of many regulatory proteins and, in the fruitfly Drosophila melanogaster, buffers genetic variation in morphogenetic pathways. Levels and patterns of genetic variation differ greatly between obligatorily outbreeding species such as fruitflies and self-fertilizing species such as the plant Arabidopsis thaliana. Also, plant development is more plastic, being coupled to environmental cues. Here we report that, in Arabidopsis accessions and recombinant inbred lines, reducing Hsp90 function produces an array of morphological phenotypes, which are dependent on underlying genetic variation. The strength and breadth of Hsp90's effects on the buffering and release of genetic variation suggests it may have an impact on evolutionary processes. We also show that Hsp90 influences morphogenetic responses to environmental cues and buffers normal development from destabilizing effects of stochastic processes. Manipulating Hsp90's buffering capacity offers a tool for harnessing cryptic genetic variation and for elucidating the interplay between genotypes, environments and stochastic events in the determination of phenotype.
Arabidopsis↗
HSP101: a key component for the acquisition of thermotolerance in plants.
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Arabidopsis↗
Increased fatty acid production in potato by engineering of acetyl-CoA carboxylase.
In contrast to oil seeds, potato (Solanum tuberosum L.) is characterized by a high amount of starch stored in the tubers. To assess the capacity for oil synthesis in potato tubers, the changes in lipid content and flux into lipid synthesis were explored in transgenic potatoes altered in carbohydrate or lipid metabolism. A strong decrease in the amount of starch observed in antisense lines for ADP-glucose pyrophosphorylase or plastidic phosphoglucomutase had no effect on storage-lipid content. Similarly, potato lines over-expressing the Arabidopsis thaliana (L.) Heynh. plastidic ATP/ADP transporter that contained an increased amount of starch were not altered in oil content, indicating that the plastidic ATP level is not limiting fatty acid synthesis in potato tubers. However, over-expression of the acetyl-CoA carboxylase from Arabidopsis in the amyloplasts of potato tubers led to an increase in fatty acid synthesis and a more than 5-fold increase in the amount of triacylglycerol. Taken together, these data demonstrate that potato tubers have the capacity for storage-lipid synthesis and that malonyl-CoA, the substrate for elongation during fatty acid synthesis, represents one of the limiting factors for oil accumulation.
Acetyl-CoA Carboxylase↗
The development of a molecular cloning system in higher plants.
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Chromosome Mapping↗
Metabolic engineering of valine- and isoleucine-derived glucosinolates in Arabidopsis expressing CYP79D2 from Cassava.
Glucosinolates are amino acid-derived natural products that, upon hydrolysis, typically release isothiocyanates with a wide range of biological activities. Glucosinolates play a role in plant defense as attractants and deterrents against herbivores and pathogens. A key step in glucosinolate biosynthesis is the conversion of amino acids to the corresponding aldoximes, which is catalyzed by cytochromes P450 belonging to the CYP79 family. Expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in Arabidopsis resulted in the production of valine (Val)- and isoleucine-derived glucosinolates not normally found in this ecotype. The transgenic lines showed no morphological phenotype, and the level of endogenous glucosinolates was not affected. The novel glucosinolates were shown to constitute up to 35% of the total glucosinolate content in mature rosette leaves and up to 48% in old leaves. Furthermore, at increased concentrations of these glucosinolates, the proportion of Val-derived glucosinolates decreased. As the isothiocyanates produced from the Val- and isoleucine-derived glucosinolates are volatile, metabolically engineered plants producing these glucosinolates have acquired novel properties with great potential for improvement of resistance to herbivorous insects and for biofumigation.
Arabidopsis↗
Hairy root-activation tagging: a high-throughput system for activation tagging in transformed hairy roots.
Activation tagging is a powerful technique for generating gain-of-function mutants in plants. We developed a new vector system for activation tagging of genes in "transformed hairy roots". The binary vector pHR-AT (Hairy Root-Activation Tagging) and its derivative pHR-AT-GFP contain a cluster of rol (rooting locus) genes together with the right border facing four tandem repeats of the cauliflower mosaic virus (CaMV) 35S enhancer element on the same T-DNA. Transformation experiments using Arabidopsis, potato, and tobacco as model plants revealed that upon inoculating plants with Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants within a short period of time, and root culture lines were subsequently established. Molecular analyses of the pHR-AT-GFP-transformed Arabidopsis lines showed that expression of the genes adjacent to the T-DNA insertion site was significantly increased. This system may facilitate application of the activation-tagging approach to plant species that are recalcitrant to the regeneration of transgenic plants. High-throughput metabolic profiling of activation-tagged root culture lines will offer opportunities for identifying regulatory or biosynthetic genes for the production of valuable secondary metabolites of interest.
Arabidopsis↗
Monitoring horizontal gene transfer.
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Ecosystem↗
Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.
Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.
Arabidopsis↗
Expression of an engineered granule-bound Escherichia coli maltose acetyltransferase in wild-type and amf potato plants.
Starch is used in many industrial applications, but often requires chemical derivatization to enhance its properties before use. In particular, the stability of starch polymers in solution is improved by acetylation. A drawback of this treatment is the use of pollutant chemicals. A biological alternative to chemical derivatization was investigated by the expression of an amyloplast-targeted Escherichia coli maltose acetyltransferase (MAT) gene in tubers of wild-type (Kardal) and mutant amylose-free (amf) potato plants. MAT was expressed as such, or fused to the N- or C-terminus of a non-catalytic starch-binding domain (SBD) to target the starch granule. Starch granules derived from transgenic plants were found to contain acetyl groups, although their content was low, opening up an avenue to move away from the post-harvest chemical derivatization of starch. MAT inside starch granules was found to be active post-harvest when supplied with acetyl-coenzyme A and glucose or maltose, but it did not acetylate starch polymers in vitro. Starch granules from transformants in which MAT alone was expressed also showed MAT activity, indicating that MAT is accumulated in starch granules, and has affinity for starch by itself. Furthermore, starch granule morphology was altered, and fusion proteins containing MAT and SBD seemed to have a higher affinity for starch granules than two appended SBDs. These results are discussed against the background of the quaternary structure of MAT.
Acetyltransferases↗
A yeast artificial chromosome-splitting vector designed for precise manipulation of specific plant chromosome region.
A yeast artificial chromosome (YAC) splitting vector, pKI01, was constructed for manipulating plant chromosome fragments cloned as YACs in order to transfer specific regions of the fragments into plant cells. Vector pKI01 consists of Km(r) and ADE2 genes (selective markers for plant and yeast transformants, respectively), inverted telomeric repeats Tr and CEN4. To demonstrate the utility of pKI01, YAC CIC9e2 harboring a 590-kb fragment from Arabidopsis thaliana chromosome 5 was split into specific fragments. A 1-kb target region positioned 100 kb from the right end of the 590 kb fragment was cloned into pKI01. The resultant plasmid, pKY03, was introduced into Saccharomyces cerevisiae harboring YAC CIC9e2. The Ade+ transformants were found to contain two new YACs of 490 and 100 kb, and to lack the original 590 kb YAC, consistent with the expected splitting event. To release the desired middle region of YAC CIC9e2, two additional splitting vectors were constructed, pKY11 and pKY14. By conducting two rounds of splitting, i.e., the first round 100 kb from the right end of YAC CIC9e2 with pKY11 to generate 490 and 100 kb YACs and a second round 50 kb from the right end of the new 490 kb YAC to generate 440 and 50 kb YACs, the middle 50 kb region of a plant chromosome fragment harboring Km(r) was successfully released as a split YAC. These results indicate that YAC splitting vectors as constructed in this study are useful for generating any desired plant chromosome fragment as a YAC for eventual re-introduction into plant cells.
Arabidopsis↗
[Genetic transformation of autotetraploid Isatis indigotica fort. induced by Ri T-DNA and plant regeneration].
OBJECTIVE: To establish an effective system for genetic transformation of autotetraploid Isatis indigotica by Agrobacterium rhizogenes. METHOD: Agrobacterium rhizogenes strains R1601, ATCC15834 and A4 were employed to induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots were developed into regenerated plants on solid MS media with different Kinds of plant growth regulators. RESULT: Root induction differed obviously. By three Agrobacterium strains. Hairy roots grew rapidly on solid MS medium without plant growth regulators and showed the typical hairy root phenotype: profuse branching, high density of root hairs and plagiotropism. And the transformation of Ri T-DNA was confirmed by opine analysis. The biomass of hairy roots increased nearly 35 times regulator-free MS after suspended in plant growth two weeks. On solid MS medium with BA, adventitious buds were differentiated directly from hairy roots without callus formation. All of the adventitious buds were rooted on root induction medium and developed into regenerated plants. Opine analysis indicated the integration of Ri T-DNA in the transformed plants. CONCLUSION: Agrobacterium rhizogene can induce hairy roots from autotetraploid Isatis indigotica, and the obtained hairy roots can develop into regenerated plants.
Coculture Techniques↗
Engineering novel carotenoids in microorganisms.
A considerable number of microbial and plant carotenoid biosynthesis genes have been cloned over the past few years. Functional heterologous expression of most of these genes has made it possible to engineer carotenoid biosynthesis in non-carotenogenic E. coli and yeasts. Recently, gene combination and molecular breeding of pathways have been used to produce novel and rare high-value carotenoids.
Bacteria↗
A promoter directing epidermal expression in transgenic alfalfa.
We describe the isolation, cloning and expression of a 2.8 kb promoter fragment of the Blec4 gene from pea (Pisum sativum cv. Alaska) and demonstrate that it is capable of directing the expression of the beta-glucuronidase coding region to the developing epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa (Medicago sativa cv. RegenSY). The Blec4 promoter represents a useful tool with which to target the expression of foreign genes to the epidermal layer of actively growing shoots. The activity of the Blec4 promoter in the epidermis of the shoot apex makes it particularly suitable for genetically engineering defense against insects and diseases that attack the growing shoot apex.
Fabaceae↗
Polygalacturonase inhibiting protein in plant cell walls
Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.
Journal Article↗