Biomedical subjects

Abhaya M Dandekar

Publications and source records attributed to Abhaya M Dandekar.

14 recordsLinked to original sources

PubMed2006

Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality.

Sorbitol and sucrose are major products of photosynthesis distributed in apple trees (Malus domestica Borkh. cv. "Greensleeves") that affect quality in fruit. Transgenic apple plants were silenced or up-regulated for sorbitol-6-phosphate dehydrogenase by using the CaMV35S promoter to define the role of sorbitol distribution in fruit development. Transgenic plants with suppressed sorbitol-6-phosphate dehydrogenase compensated by accumulating sucrose and starch in leaves, and morning and midday net carbon assimilation rates were significantly lower. The sorbitol to sucrose ratio in leaves was reduced by approximately 90% and in phloem exudates by approximately 75%. The fruit accumulated more glucose and less fructose, starch, and malic acid, with no overall differences in weight and firmness. Sorbitol dehydrogenase activity was reduced in silenced fruit, but activities of neutral invertase, vacuolar invertase, cell wall-bound invertase, fructose kinase, and hexokinase were unaffected. Analyses of transcript levels and activity of enzymes involved in carbohydrate metabolism throughout fruit development revealed significant differences in pathways related to sorbitol transport and breakdown. Together, these results suggest that sorbitol distribution plays a key role in fruit carbon metabolism and affects quality attributes such as sugar-acid balance and starch accumulation.

Fruit↗

PubMed2006

Jug r 4, a legumin group food allergen from walnut (Juglans regia Cv. Chandler).

Allergy to walnut is the most frequently reported tree nut allergy in the United States. Walnut 2S albumin, a vicilin-like protein, and a lipid transfer protein allergen have previously been described. Our objective was to clone and express a cDNA encoding a legumin group protein, assess IgE-binding with sera from walnut allergic patients, and investigate cross-reactivity with selected nuts. Primers were used to obtain the cDNA by 5' and 3' rapid amplification of cDNA ends from walnut mRNA. The cDNA was subcloned into the pMAL-c2X vector and the recombinant fusion protein, named rJug r 4, was expressed in Escherichia coli. The obtained cDNA encoded a precursor protein with a predicted molecular weight of 58.1 kD, which showed significant sequence homology to hazelnut and cashew legumin allergens. Serum IgE from 21 of 37 (57%) patients bound the rJug r 4 fusion protein. In vitro cross-reactivity was demonstrated with hazelnut, cashew, and peanut protein extracts.

Allergens↗

PubMed2006

Down-regulation of sorbitol dehydrogenase and up-regulation of sucrose synthase in shoot tips of the transgenic apple trees with decreased sorbitol synthesis.

Both sorbitol and sucrose are translocated to, and utilized in, sink tissues of apple (Malus domestica). Considering that antisense suppression of aldose 6-phosphate reductase resulted in lower concentrations of sorbitol and higher concentrations of sucrose in source leaves without altering the vegetative growth of apple trees, it was hypothesized that sorbitol metabolism is down-regulated and sucrose metabolism is up-regulated in shoot tips of the transgenic plants. Carbohydrate measurements indicated that sorbitol concentration was lower whereas sucrose concentration was higher in the shoot tips of transgenic apple plants with decreased sorbitol synthesis compared with the untransformed control. However, the shoot relative growth rate was not altered in the transgenic plants. Sorbitol dehydrogenase (SDH) activity was decreased; acid invertase activity and neutral invertase activity remained the same, whereas sucrose synthase (SUSY) activity was increased in shoot tips of the transgenic plants. The SDH transcript level was lower whereas the SUSY transcript level was higher in shoot tips of the transgenic plants. SDH activity and SDH transcript level were specifically stimulated by exogenous sorbitol fed to the shoot tips via the transpiration stream but were specifically inhibited by sucrose. SUSY activity and SUSY transcript level were dramatically enhanced by sucrose, but decreased by glucose and fructose. Neither acid invertase nor neutral invertase activity responded to sucrose, glucose, fructose, or any other sugars tested. It is concluded that sorbitol dehydrogenase is down-regulated, whereas sucrose synthase is up-regulated in shoot tips of the transgenic apple trees with decreased sorbitol synthesis, leading to homeostasis of vegetative growth. Sorbitol and sucrose act as signal molecules to modulate the expression and activities of sorbitol dehydrogenase and sucrose synthase, both of which play an important role in determining the sink strength of apple shoot tips.

Down-Regulation↗

PubMed2006

Apple (Malus x domestica).

Apple (Malus x domestica) is one of the most consumed fruit crops in the world. The major production areas are the temperate regions, however, because of its excellent storage capacity it is transported to distant markets covering the four corners of the earth. Transformation is a key to sustaining this demand - permitting the potential enhancement of existing cultivars as well as to investigate the development of new cultivars resistant to pest, disease, and storage problems that occur in the major production areas. In this paper we describe an efficient Agrobacterium tumefaciens-mediated transformation protocol that utilizes leaf tissues from in vitro grown plants. Shoot regeneration is selected with kanamycin using the selectable kanamycin phosphotransferase (APH(3)II) gene and the resulting transformants confirmed using the scorable uidA gene encoding the bacterial beta-glucuronidase (GUS) enzyme via histochemical staining. Transformed shoots are propagated, rooted to create transgenic plants that are then introduced into soil, acclimatized and transferred to the greenhouse from where they are taken out into the orchard for field-testing.

Agrobacterium tumefaciens↗

PubMed2006

Walnut (Juglans).

Walnut species are important nut and timber producers in temperate regions of Europe, Asia, South America, and North America. Trees can be impacted by Phytophthora, crown gall, nematodes, and cherry leaf roll virus; nuts can be severely damaged by codling moth and Xanthomonas blight. The long-generation time of walnuts and an absence of identified natural resistance for most of these problems suggest biotechnological approaches to crop improvement. Described here is a somatic embryo based transformation protocol that has been used to successfully insert horticulturally useful traits into walnut. Selection is based on the combined use of the selectable neomycin phosphotransferase (nptII) gene and the scorable uidA gene. Transformed embryos can be germinated or micropropagated and rooted for plant production. The method described has been used to establish field trials of mature trees.

Agrobacterium tumefaciens↗

PubMed2005

Relationship of ethylene biosynthesis to volatile production, related enzymes, and precursor availability in apple peel and flesh tissues.

Regulation of ethylene biosynthesis or action has a major effect on volatiles production in apples. To understand the biochemical processes involved, we used Greensleeves apples from a transgenic line with a high suppression of ethylene biosynthesis. The study was focused at the level of the aroma volatile-related enzymes, including alcohol acyltransferase (AAT), alcohol dehydrogenase (ADH), and lipoxygenase (LOX) and at the level of amino acids and fatty acids as aroma volatile precursors in peel and flesh tissues. In general, volatile production, enzyme activity levels, and precursor availability were higher in the peel than the flesh and were differentially affected by ethylene regulation. AAT enzyme activity showed a clear pattern concomitant with ethylene regulation. Contrarily, ADH and LOX seem to be independent of ethylene modulation. Isoleucine, an important precursor of aroma compounds including 2-methylbutanoate esters, showed a major increase in the peel during ripening and responded significantly to ethylene regulation. Other important aroma volatiles precursors, like linoleic and linolenic acid, showed an accumulation during ripening associated with increases in aldehydes. The significance of these changes in relation to aroma volatile production is discussed.

Acyltransferases↗

PubMed2005

Plant transformation: agrobacterium-mediated gene transfer.

Plant transformation is the process by which DNA is introduced into plant cells or tissues. The DNA can come from virtually any source. Gene transfer methodology has become part of an essential technology to manipulate plants for both scientific and commercial purposes. Transgenic plants, the products of this methodology, are useful for dissecting the mechanism(s) of plant gene regulation. This technology is also useful for identifying and evaluating agriculturally useful traits (genes) as well as for their introduction into commercially valuable crops. One of the most efficient methods for gene transfer employs Agrobacterium tumefaciens and takes advantage of the naturally evolved crown gall-inducing mechanisms of DNA transfer present in this common soil pathogen. Much has been learned about the mechanisms of this form of DNA movement and subsequent crown gall induction. This information has been applied to develop methods that result in the formation of gall-free, genetically transformed plants. This chapter describes a detailed protocol for Agrobacterium-mediated transformation of tobacco cells and their subsequent selection and regeneration into transgenic plants.

Gene Transfer Techniques↗

PubMed2005

Electroporation: introduction and expression of transgenes in plant protoplasts.

An optimized protocol for the electroporation-based transfection of tobacco protoplasts is described that routinely results in transgene expression frequencies approaching 90%. The overall efficiency of the procedure depends collectively on numerous key parameters, including protoplast viability; DNA concentration, purity, and topology; carrier DNA; and electrical conditions such as ionic strength of the electroporation buffer, electric field strength, pulse duration, and capacitance. Individual methodologies that address each one of these parameters are presented in sufficient detail to enable successful reproduction of this method along with notes that describe helpful tips.

Electroporation↗

PubMed2004

Antisense inhibition of sorbitol synthesis leads to up-regulation of starch synthesis without altering CO2 assimilation in apple leaves.

Sorbitol is a primary end-product of photosynthesis in apple (Malus domestica Borkh.) and many other tree fruit species of the Rosaceae family. Sorbitol synthesis shares a common hexose phosphate pool with sucrose synthesis in the cytosol. In this study, 'Greensleeves' apple was transformed with a cDNA encoding aldose 6-phosphate reductase (A6PR, EC 1.1.1.200) in the antisense orientation. Antisense expression of A6PR decreased A6PR activity in mature leaves to approximately 15-30% of the untransformed control. The antisense plants had lower concentrations of sorbitol but higher concentrations of sucrose and starch in mature leaves at both dusk and predawn. (14)CO(2) pulse-chase labeling at ambient CO(2) demonstrated that partitioning of the newly fixed carbon to starch was significantly increased, whereas that to sucrose remained unchanged in the antisense lines with decreased sorbitol synthesis. Total activities of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), sucrose-phosphate synthase (EC 2.4.1.14), and ADP-glucose pyrophosphorylase (EC 2.7.7.27) were not significantly altered in the antisense lines, whereas both stromal and cytosolic fructose-1,6-bisphosphatase (EC 3.1.3.11) activities were higher in the antisense lines with 15% of the control A6PR activity. Concentrations of glucose 6-phosphate and fructose 6-phosphate (F6P) were higher in the antisense plants than in the control, but the 3-phosphoglycerate concentration was lower in the antisense plants with 15% of the control A6PR activity. Fructose 2, 6-bisphosphate concentration increased in the antisense plants, but not to the extent expected from the increase in F6P, comparing sucrose-synthesizing species. There was no significant difference in CO(2) assimilation in response to photon flux density or intercellular CO(2) concentration. We concluded that cytosolic FBPase activity in vivo was down-regulated and starch synthesis was up-regulated in response to decreased sorbitol synthesis. As a result, CO(2) assimilation in source leaves was sustained at both ambient CO(2) and saturating CO(2).

Carbon Dioxide↗

PubMed2004

Impact of suppression of ethylene action or biosynthesis on flavor metabolites in apple (Malus domestica Borkh) fruits.

To understand the role of ethylene in regulating the overall flavor of apple fruits, ethylene production or action was reduced using transgenic apple trees suppressed for ACC-synthase or ACC-oxidase enzyme activity or by the addition of 1-methylcyclopropene (1-MCP), an ethylene action inhibitor. Flavor components were differentially regulated in response to the suppression of both ethylene biosynthesis and action. Headspace analysis of aroma production, an ethylene-associated event, showed a reduction in ester and alcohol production in the ethylene-suppressed lines and in the apples treated with 1 microL L(-1) 1-MCP for 20 h at 20 degrees C. However, no major differences were observed in the concentrations of aldehyde volatiles. Other flavor metabolites that showed an ethylene-dependent pattern were organic acids and sugars. Malic acid degradation was significantly reduced under ethylene-suppressed conditions, showing a recovery after the fruit was exposed to ethylene. Sucrose and fructose concentrations were influenced by suppression or enhancement of ethylene. Total phenolics as well as individual phenolic compounds showed an ethylene-dependent regulation only in response to the suppression of ethylene biosynthesis, but not when ethylene action was inhibited.

Amino Acid Oxidoreductases↗

PubMed2004

The chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 degrees C.

Since, like other osmolytes, proline can act as a protein stabilizer, we investigated the thermoprotectant properties of proline in vitro and in vivo. In vivo, elevated proline pools in Escherichia coli (obtained by altering the feedback inhibition by proline of gamma-glutamylkinase, the first enzyme of the proline biosynthesis pathway) restore the viability of a dnaK-deficient mutant at 42 degrees C, suggesting that proline can act as a thermoprotectant for E. coli cells. Furthermore, analysis of aggregated proteins in the dnaK-deficient strain at 42 degrees C by two-dimensional gel electrophoresis shows that high proline pools reduce the protein aggregation defect of the dnaK-deficient strain. In vitro, like other "chemical chaperones," and like the DnaK chaperone, proline protects citrate synthase against thermodenaturation and stimulates citrate synthase renaturation after urea denaturation. These results show that a protein aggregation defect can be compensated for by a single mutation in an amino acid biosynthetic pathway and that an ubiquitously producible chemical chaperone can compensate for a defect in one of the major chaperones involved in protein folding and aggregation.

Citrate (si)-Synthase↗

PubMed2003

Agrobacterium tumefaciens as an agent of disease.

Twenty-six years ago it was found that the common soil bacterium Agrobacterium tumefaciens is capable of extraordinary feats of interkingdom genetic transfer. Since this discovery, A. tumefaciens has served as a model system for the study of type IV bacterial secretory systems, horizontal gene transfer and bacterial-plant signal exchange. It has also been modified for controlled genetic transformation of plants, a core technology of plant molecular biology. These areas have often overshadowed its role as a serious, widespread phytopathogen - the primary driver of the first 80 years of Agrobacterium research. Now, the diverse areas of A. tumefaciens research are again converging because new discoveries in transformation biology and the use of A. tumefaciens vectors are allowing the development of novel, effective biotechnology-based strategies for the control of crown gall disease.

Agrobacterium tumefaciens↗

PubMed2003

Structural and transcriptional analysis of the self-incompatibility locus of almond: identification of a pollen-expressed F-box gene with haplotype-specific polymorphism.

Gametophytic self-incompatibility in Rosaceae, Solanaceae, and Scrophulariaceae is controlled by the S locus, which consists of an S-RNase gene and an unidentified "pollen S" gene. An approximately 70-kb segment of the S locus of the rosaceous species almond, the S haplotype-specific region containing the S-RNase gene, was sequenced completely. This region was found to contain two pollen-expressed F-box genes that are likely candidates for pollen S genes. One of them, named SFB (S haplotype-specific F-box protein), was expressed specifically in pollen and showed a high level of S haplotype-specific sequence polymorphism, comparable to that of the S-RNases. The other is unlikely to determine the S specificity of pollen because it showed little allelic sequence polymorphism and was expressed also in pistil. Three other S haplotypes were cloned, and the pollen-expressed genes were physically mapped. In all four cases, SFBs were linked physically to the S-RNase genes and were located at the S haplotype-specific region, where recombination is believed to be suppressed, suggesting that the two genes are inherited as a unit. These features are consistent with the hypothesis that SFB is the pollen S gene. This hypothesis predicts the involvement of the ubiquitin/26S proteasome proteolytic pathway in the RNase-based gametophytic self-incompatibility system.

Amino Acid Sequence↗

PubMed2002

Expression of recombinant trichosanthin, a ribosome-inactivating protein, in transgenic tobacco.

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.

Cucumovirus↗