Genetic Enhancement of Cold Tolerance by Expression of a Gene for Chloroplast [omega]-3 Fatty Acid Desaturase in Transgenic Tobacco

The increased production of trienoic fatty acids, hexadecatrienoic (16:3) and linolenic (18:3) acids, is a response connected with cold acclimation of higher plants and is thought to protect plant cells against cold damage. Transgenic tobacco (Nicotiana tabacum cv SR1) plants that contain increased levels of 16:3 and 18:3 fatty acids, and correspondingly decreased levels of their precursors, hexadecadienoic and linoleic acids, were engineered by introduction of a chloroplast [omega]-3 fatty acid desaturase gene (the fad7 gene) isolated from Arabidopsis thaliana. When exposed to 1[deg]C for 7 d and then cultured at 25[deg]C, the suppression of leaf growth observed in the wild-type plants was significantly alleviated in the transgenic plants with the fad7 gene. The low-temperature- induced chlorosis was also much reduced in the plants transformed with the fad7 gene. These results indicate that increased levels of trienoic fatty acids in genetically engineered plants enhance cold tolerance.     

EF-1[alpha] Is Associated with a Cytoskeletal Network Surrounding Protein Bodies in Maize Endosperm Cells

By using indirect immunofluorescence and confocal microscopy, we documented changes in the distribution of elongation factor-1[alpha] (EF-1[alpha]), actin, and microtubules during the development of maize endosperm cells. In older interphase cells actively forming starch grains and protein bodies, the protein bodies are enmeshed in EF-1[alpha] and actin and are found juxtaposed with a multidirectional array of microtubules. Actin and EF-1[alpha] appear to exist in a complex, because we observed that the two are colocalized, and treatment with cytochalasin D resulted in the redistribution of EF-1[alpa]. These data suggest that EF-1[alpha] and actin are associated in maize endosperm cells and may help to explain the basis of the correlation we found between the concentration of EF-1[alpha] and lysine content. The data also support the hypothesis that the cytoskeleton plays a role in storage protein deposition. The distributions of EF-1[alpha] actin, and microtubules change during development. We observed that in young cells before the accumulation of starch and storage protein, EF-1[alpha], actin, and microtubules are found mainly in the cell cortex or in association with nuclei.     

Endosperm Protein Synthesis and l-[S]Methionine Incorporation in Maize Kernels Cultured In Vitro

This study was conducted to examine protein synthesis and l-[³⁵S] methionine incorporation into the endosperm of Zea mays L. kernels developing in vitro. Two-day-old kernels of the inbred line W64A were placed in culture on a defined medium containing 10 microCuries l-[³⁵S] methionine per milliliter (13 milliCuries per millimole) and harvested at 10, 15, 20, 25, 30, 35, and 40 days after pollination. Cultured kernels attained a final endosperm mass of 120 milligrams compared to 175 milligrams for field-grown controls. Field and cultured kernels had similar concentrations (microgram per milligram endospern) for total protein, albumin plus globulin, zein, and glutelin fractions at most kernel ages.     

Class I [beta]-1,3-Glucanases in the Endosperm of Tobacco during Germination

Rupture of the seed coat and rupture of the endosperm are separate events in the germination of Nicotiana tabacum L. cv Havana 425 seeds. Treatment with 10-5 M abscisic acid (ABA) did not appreciably affect seed-coat rupture but greatly delayed subsequent endosperm rupture by more than 100 h and resulted in the formation of a novel structure consisting of the enlarging radicle with a sheath of greatly elongated endosperm tissue. Therefore, ABA appears to act primarily by delaying endosperm rupture and radicle emergence. Measurements of [beta]-1,3-glucanase activity, antigen content, and mRNA accumulation together with reporter gene experiments showed that induction of class I [beta]-1,3-glucanase genes begins just prior to the onset of endosperm rupture but after the completion of seed-coat rupture. This induction was localized exclusively in the micropylar region of the endosperm, where the radicle will penetrate. ABA treatment markedly inhibited the rate of [beta]-1,3-glucanase accumulation but did not delay the onset of induction. Independent of the ABA concentration used, onset of endosperm rupture was correlated with the same [beta]-1,3-glucanase content/seed. These results suggest that ABA-sensitive class I [beta]-1,3-glucanases promote radicle penetration of the endosperm, which is a key limiting step in tobacco seed germination.     

Abscisic Acid Stimulated Osmotic Adjustment and Its Involvement in Adaptation of Tobacco Cells to NaCl

Osmotic adjustment of cultured tobacco (Nicotiana tabacum L. var Wisconsin 38) cells was stimulated by 10 micromolar (±) abscisic acid (ABA) during adaptation to water deficit imposed by various solutes including NaCl, KCl, K₂SO₄, Na₂SO₄, sucrose, mannitol, or glucose. The maximum difference in cell osmotic potential (Ψπ) caused by ABA treatment during adaptation to 171 millimolar NaCl was about 6 to 7 bar. The cell Ψπ differences elicited by ABA were not due to growth inhibition since ABA stimulated growth of cells in the presence of 171 millimolar NaCl. ABA caused a cell Ψπ difference of about 1 to 2 bar in medium without added NaCl. Intracellular concentrations of Na⁺, K⁺, Cl⁻, free amino acids, or organic acids could not account for the Ψπ differences induced by ABA in NaCl treated cells. However, since growth of NaCl treated cells is more rapid in the presence of ABA than in its absence, greater accumulation of Na⁺, K⁺, and Cl⁻ was necessary for ion pool maintenance. Higher intracellular sucrose and reducing sugar concentrations could account for the majority of the greater osmotic adjustment of ABA treated cells. More rapid accumulation of proline associated with ABA treatment was highly correlated with the effects of ABA on cell Ψπ. These and other data indicate that the role of ABA in accelerating salt adaptation is not mediated by simply stimulating osmotic adjustment.     

The 5[prime] Flanking Regions of Vicilin and Napin Storage Protein Genes Are Down-Regulated by Desiccation in Transgenic Tobacco

Drying of seeds, when imposed prematurely, elicits a switch in metabolism; events unique to development, such as synthesis of storage protein, are terminated, whereas syntheses associated with germination and growth are initiated. To determine the role of desiccation in down-regulating the expression of genes for storage proteins, the desiccation responsiveness of the 5[prime] and 3[prime] regulatory regions of the genes encoding the pea storage protein vicilin and the Brassica napus storage protein napin was tested in transgenic tobacco seed. Chimeric genes were introduced into tobacco; these genes consisted of the coding region of the reporter gene for [beta]-glucuronidase (GUS) and 5[prime] and/or 3[prime] regions from the vicilin or napin genes or, as controls, the same regions derived from constitutively expressed genes, presumed to be desiccation insensitive. In transgenic seed expressing the gene constructs containing the vicilin or napin promoters, GUS activities declined during late seed development, and more dramatically, after imbibition of mature dry seed or prematurely dried seed. In contrast, GUS activities increased after seed rehydration when the constitutive viral promoter replaced the storage-protein gene 5[prime] region. Transient expression assays support the hypothesis that premature drying down-regulates the expression of the storage-protein gene promoter. Following desiccation, this region may become insensitive to positive controlling factors; alternatively, changes to trans-acting factors may occur.     

农杆菌介导番茄铁载体蛋白基因(LeIRT2)转化烟草的研究

构建了含有目的基因(LeIRT2)的双元载体pYF840,并成功地将植物性内含子构建到筛选标记基因新霉素磷酸转移酶基因(nptⅡ)和gus报告基因中。利用农杆菌介导法,将铁载体蛋白(LeIRT2)基因导入革新一号烟草,PCR、PCR-Southern blotting及Southern blottlng检测结果显示,有3个烟草株系的基因组中整合了完整的铁载体基因(LeIRT2)。水培试验结果显示转基因株系1、3不但提高了植株的铁吸收能力,而且还促进了植株的生长,但转基因株系2却与对照无明显区别。     

Heme Inhibition of [delta]-Aminolevulinic Acid Synthesis Is Enhanced by Glutathione in Cell-Free Extracts of Chlorella

In plants, algae, and many bacteria, the heme and chlorophyll precursor, [delta]-aminolevulinic acid (ALA), is synthesized from glutamate in a reaction involving a glutamyl-tRNA intermediate and requiring ATP and NADPH as cofactors. In particulate-free extracts of algae and chloroplasts, ALA synthesis is inhibited by heme. Inclusion of 1.0 mM glutathione (GSH) in an enzyme and tRNA extract, derived from the green alga Chlorella vulgaris, lowered the concentration of heme required for 50% inhibition approximately 10-fold. The effect of GSH could not be duplicated with other reduced sulfhydryl compounds, including mercaptoethanol, dithiothreitol, and cysteine, or with imidazole or bovine serum albumin, which bind to heme and dissociate heme dimers. Absorption spectroscopy indicated that heme was fully reduced in incubation medium containing dithiothreitol, and addition of GSH did not alter the heme reduction state. Oxidized GSH was as effective in enhancing heme inhibition as the reduced form. Co-protoporphyrin IX inhibited ALA synthesis nearly as effectively as heme, and 1.0 mM GSH lowered the concentration required for 50% inhibition approximately 10-fold. Because GSH did not influence the reduction state of heme in the incubation medium, and because GSH could not be replaced by other reduced sulfhydryl compounds or ascorbate, the effect of GSH cannot be explained by action as a sulfhydryl protectant or heme reductant. Preincubation of enzyme extract with GSH, followed by rapid gel filtration, could not substitute for inclusion of GSH with heme during the reaction. The results suggest that GSH must specifically interact with the enzyme extract in the presence of the inhibitor to enhance the inhibition.     

Ribonucleic Acid Regulation in Permeabilized Cells of Escherichia coli Capable of Ribonucleic Acid and Protein Synthesis

A cell permeabilization procedure is described that reduces viability less than 10% and does not significantly reduce the rates of ribonucleic acid and protein synthesis when appropriately supplemented. Permeabilization abolishes the normal stringent coupling of protein and ribonucleic acid synthesis.     

Evidence That a Malate/Inorganic Phosphate Exchange Translocator Imports Carbon across the Leucoplast Envelope for Fatty Acid Synthesis in Developing Castor Seed Endosperm

In this study we examined the processes by which malate and pyruvate are taken up across the leucoplast envelope for fatty acid synthesis in developing castor (Ricinus communis L.) seed endosperm. Malate was taken up by isolated leucoplasts with a concentration dependence indicative of protein-mediated transport. The maximum rate of malate uptake was 704 [plus or minus] 41 nmol mg-1 protein h-1 and the Km was 0.62 [plus or minus] 0.08 mM. In contrast, the rate of pyruvate uptake increased linearly with respect to the substrate concentration and was 5-fold less than malate at a concentration of 5 mM. Malate uptake was inhibited by inorganic phosphate (Pi), glutamate, malonate, succinate, 2-oxoglutarate, and n-butyl malonate, an inhibitor of the mitochondrial malate/Pi-exchange translocator. Back-exchange experiments confirmed that malate was taken up by leucoplasts in counterexchange for Pi. The exchange stoichiometry was 1:1. The rate of malate-dependent fatty acid synthesis by isolated leucoplasts was 3-fold greater than from pyruvate at a concentration of 5 mM and was inhibited by n-butyl malonate. It is proposed that leucoplasts from developing castor endosperm contain a malate/Pi translocator that imports malate for fatty acid synthesis. This type of dicarboxylate transport activity has not been identified previously in plastids.     

Overexpression of [delta]-Pyrroline-5-Carboxylate Synthetase Increases Proline Production and Confers Osmotolerance in Transgenic Plants

Proline (Pro) accumulation has been correlated with tolerance to drought and salinity stresses in plants. Therefore, overproduction of Pro in plants may lead to increased tolerance against these abiotic stresses. To test this possibility, we overexpressed in tobacco the mothbean [delta]-pyrroline-5-carboxylate synthetase, a bifunctional enzyme able to catalyze the conversion of glutamate to [delta]-pyrroline-5-carboxylate, which is then reduced to Pro. The transgenic plants produced a high level of the enzyme and synthesized 10- to 18-fold more Pro than control plants. These results suggest that activity of the first enzyme of the pathway is the rate-limiting factor in Pro synthesis. Exogenous supply of nitrogen further enhanced Pro production. The osmotic potentials of leaf sap from transgenic plants were less decreased under water-stress conditions compared to those of control plants. Overproduction of Pro also enhanced root biomass and flower development in transgenic plants under drought-stress conditions. These data demonstrated that Pro acts as an osmoprotectant and that overproduction of Pro results in the increased tolerance to osmotic stress in plants.     

天麻抗真菌蛋白基因的克隆及其启动子活性

通过构建和筛选天麻（Gastrodia elata Bl)基因组文库,克隆了一个天麻抗真菌蛋白基因组DNA。该基因组DNA含有一个516碱基组成的编码区。没有内含子结构。其启动子区含有保守的TATA盒及CAAT盒。为研究启动子活性。构建了-1157bp启动子区与GUS基因的融合表达载体。并将其用农杆菌（Agrobacterium tumefaciens)介导的遗传转化方法导入烟草（Nicotiana tabacum)中,获得了稳定转化的烟草。利用荧光检测及组织化学染色法对GUS表达进行了分析。结果表明,该启动子能够启动GUS基因在转基因烟草中组织特异性地表达。GUS基因在根中的表达水平最高。茎中次之,叶中只有低水平表达,而且该启动子具有诱导表达活性。可被真菌及水杨酸,茉莉酸强烈诱导表达。     

天麻抗真菌蛋白基因的克隆及其启动子活性

通过构建和筛选天麻(Gastrodia elata Bl.)基因组文库,克隆了一个天麻抗真菌蛋白基因组DNA.该基因组DNA含有一个516碱基组成的编码区,没有内含子结构.其启动子区含有保守的TATA盒及CAAT盒.为研究启动子活性,构建了-1 157 bp启动子区与GUS基因的融合表达载体.并将其用农杆菌(Agrobacterium tumefaciens)介导的遗传转化方法导入烟草(Nicotiana tabacum)中,获得了稳定转化的烟草.利用荧光检测及组织化学染色法对GUS表达进行了分析.结果表明,该启动子能够启动GUS基因在转基因烟草中组织特异性地表达.GUS基因在根中的表达水平最高,茎中次之,叶中只有低水平表达.而且该启动子具有诱导表达活性,可被真菌及水杨酸、茉莉酸强烈诱导表达.     

Synthesis of Glutathione in Leaves of Transgenic Poplar Overexpressing [gamma]-Glutamylcysteine Synthetase

Internode stem fragments of the poplar hybrid Populus tremula x Populus alba were transformed with a bacterial gene (gshl) for [gamma]-glutamylcysteine synthetase ([gamma]-ECS) targeted to the cytosol. Lines overexpressing [gamma]-ECS were identified by northern analysis, and the transformant with the highest enzyme activity was used to investigate the control of glutathione synthesis. Whereas foliar [gamma]-ECS activity was below the limit of detection in untransformed plants, activities of up to 8.7 nmol mg-1 protein min-1 were found in the transformant, in which the foliar contents of [gamma]-glutamylcysteine ([gamma]-EC) and glutathione were increased approximately 10- and 3-fold, respectively, without affecting either the reduction state of the glutathione pool or the foliar cysteine content. A supply of exogenous cysteine to leaf discs increased the glutathione content from both transformed and untransformed poplars, and caused the [gamma]-EC content of the transformant discs to increase still further. The following conclusions are drawn: (a) the native [gamma]-ECS of untransformed poplars exists in quantities that are limiting for foliar glutathione synthesis; (b) foliar glutathione synthesis in untransformed poplars is limited by cysteine availability; (c) in the transformant interactions between glutathione synthesis and cysteine synthesis operate to sustain the increased formation of [gamma]-EC and glutathione; and (d) the foliar glutathione content of the transformant is restricted by cysteine availability and by the activity of glutathione synthetase.     

Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase

Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). M?ssbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.     

A Short Peptide in Rice Glutelin Directs Trafficking of Protein into the Protein Storage Vacuoles of the Endosperm Cells

Several protein vacuolar sorting determinants (VSDs) have been identified in higher plants. Glutelin as a major storage protein in rice endosperm cells is transported to a protein storage vacuole (PSV). How glutelin sort to PSV and the mechanism of the intracellular trafficking has remained unknown. Here, a sequence-specific vacuolar sorting determinant (ssVSD) is identified by serial deletions of rice glutelin and its role in the protein-sorting process analyzed by transgenic approaches and transient assays. The ssVSD consists of six residues (QRLKHN) within the β-subunit of glutelin is sufficient to direct the glutelin to the protein body II in the rice endosperm cells. We found that protein-sorting via the ssVSD takes place by a ～680-kDa sorting complex containing the receptor Oryza sativa receptor-like membrane Ring-H2 3 (OsRMR3). Further study indicated that OsRMR3 and the ssVSD are essential for glutelin trafficking. Furthermore, site-directed mutagenesis showed that the leucine residues in the ssVSD are critical for protein sorting.     

Inhibition of Host Deoxyribonucleic Acid Synthesis by T4 Bacteriophage in the Absence of Protein Synthesis

The requirement for phage protein synthesis for the inhibition of host deoxyribonucleic acid synthesis has been investigated by using a phage mutant unable to catalyze the production of any phage deoxyribonucleic acid. It has been concluded that the major pathway whereby phage inhibit host syntheses requires protein synthesis. The inhibition of host syntheses by phage ghosts is not affected by inhibitors of protein synthesis.