A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme

A starch deficient mutant of Arabidopsis thaliana (L.) Heynh. has been isolated in which leaf extracts contain only about 5% as much activity of ADPglucose pyrophosphorylase (EC 2.7.7.27) as the wild type. A single, nuclear mutation at a previously undescribed locus designated adg2 is responsible for the mutant phenotype. Although the mutant contained only 5% as much ADPglucose pyrophosphorylase activity as the wild type, it accumulated 40% as much starch when grown in a 12 hour photoperiod. The mutant also contained about 40% as much starch as the wild type when grown in continuous light, suggesting that the rate of synthesis regulates its steady state accumulation. Immunological analysis of leaf extracts using antibodies against the spinach 54 and 51 kilodalton (kD) ADPglucose pyrophosphorylase subunits indicated that the mutant is deficient in a cross-reactive 54 kD polypeptide and has only about 4% as much as the wild type of a cross-reactive 51 kD polypeptide. This result and genetic studies suggested that adg2 is a structural gene which codes for the 54 kD polypeptide, and provides the first functional evidence that the 54 kD polypeptide is a required component of the native ADPglucose pyrophosphorylase enzyme.     

Role of ethylene and cytokinins in the initiation of lateral shoot growth in bromeliads

Aechmea victoriana var discolor L. B. Foster and Aechmea dactylina Bal. are commercially propagated in vitro through lateral shoot growth. A modified Murashige and Skoog medium is used which contains both BA and IAA. These growth substances were shown in the present study to synergistically stimulate the production of ethylene by the cultured plants. The stimulation of ethylene production is correlated with the outgrowth of the lateral buds. The rise in ethylene production was concluded to induce lateral shoot growth, because: (a) outgrowth of the shoots was blocked by preventing an increase in ethylene production, (b) 1-aminocyclopropane-1-carboxylic acid (ACC), the natural precursor of ethylene biosynthesis, substituted for IAA in the promotion of ethylene production and lateral bud outgrowth. Although ACC could substitute for IAA, it could not substitute for BA; therefore, cytokinins are concluded to be essential for lateral bud outgrowth in vitro in Aechmea. These results suggest that cytokinins and ethylene both play roles in natural lateral bud initiation and that the cytokinin function involves two stages of the process.     

Characterization of an HSP70 Cognate Gene Family in Arabidopsis

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5′ regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.     

Gene Expression in Developing Zea mays Embryos: Regulation by Abscisic Acid of a Highly Phosphorylated 23- to 25-kD Group of Proteins

We have earlier identified a set of proteins of 23 to 25 kilodaltons (kD), covering an isoelectric point (pI) range of 6.2 to 8.2, which accumulate gradually during normal embryogenesis of Zea mays and disappear in early germination. These polypeptides can be induced prematurely in immature embryos by abscisic acid (ABA) treatment. We report here that the more acidic protein forms are due to post-translational phosphorylation of at least two polypeptides of 23 kD, pI 8.2 and 25 kD, pI 8.0. A polyclonal antiserum was obtained which recognizes all forms of both the 23-kD and 25-kD polypeptides. Recovery of cDNA clones corresponding to these proteins was accomplished by hybridization with cDNA made from size-selected mRNA enriched for these sequences. Hybrid selection experiments demonstrate that clone MA12 specifically hybridizes with mRNAs encoding the 23-kD and 25-kD protein set which are recognized by the antiserum. By Northern hybridization analysis, the RNA encoded by clone MA12 is shown to accumulate in mature embryos and to be induced in young embryos upon ABA incubation.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

The flea ovary: ultrastructure and analysis of cell clusters

Panoistic ovarioles are found in the order of fleas (Siphonaptera). Only in some species of the Hystrichopsylloidea do polytrophic meroistic ovaries occur. No stem cells and no dividing cystocytes are found in female imagines of Hystrichopsylla talpae. However, each germ cell cluster consists of 32 cells which are generated by five mitotic cycles during the pupal stage. One of the cells containing five intercellular bridges becomes the oocyte, the others serve as nurse cells. Thus, germ cell cluster formation follows the 2(n)-rule. However, no polyfusome is found and nurse cells do not form a rosette. Furthermore, nurse cells remain small and show the same ultrastructural characters as the oocytes, which became distinguishable from nurse cells only by their enhanced growth during pre-vitellogenesis. The first phase of pre-vitellogenesis is dominated by the production of an unknown cytoplasmatic component, consisting of spherical particles, clearly distinguishable from ribosomes by diameter and contrast. The next phase is characterized by a tremendous increase in the production of ribosomes. During this second phase another cytoplasmic component consisting of ball-like structures becomes prominent. During pre-vitellogenesis, germ cell nuclei undergo a pronounced structural change in which, finally, numerous extranucleolar particles predominate. Thus, H. talpae has a polytrophic meroistic ovary, but its oocyte genomes behave panoistically.     

金缕梅科(广义)的叶结构及分类

金缕梅科(广义)Hamamelidaceae包括29属,约140种。 此科的化石出现得较早,是双子叶植物中的一个比较古老的科。 本文的目的是系统研究此科透明叶的各种性状特征,并检验它们是否具有分类学意义。 本文研究了金缕梅科29属、60余种的叶的各种性状在科内的分布及演化趋势,并根据这方面的证据对此科的分类提出一些修正意见,系统描述部分介绍了金缕梅科各属叶的形态特征。     

An in vitro test for measuring cytotoxicity of mercuric chloride to human kidney epithelial cells by specific enzyme release

Mercuric compound toxicity is well documented in animals and man for practically all organs. The recent development of cell culture techniques appeared as a novel fruitful tool in toxicology, especially in renal toxicology. Heavy metal induced renal cell alterations can be evaluated by membrane permeability damages.The present study evaluates mercuric chloride nephrotoxic effect in human kidney epithelial cells by measuring the release of two specific nephrotoxicity marker enzymes, Gamma Glutamyl Transferase (GGT) and Alkaline Phosphatase (ALP) in the culture medium. Cultured kidney epithelial cells were exposed to different HgCl₂ concentrations (5, 10, 20, and 50 g). Cultures were examined after 6 and 24 hours exposure. A good correlation between mercury dose and toxic effect, and exposure time and toxic effect was found. Enzymes were significantly released into the culture medium for 5 g and 10 g HgCl₂/ml after 6 hours exposure; and after 24 hours exposure, enzymes were released for 5 g/ml only.It appears that the specific tubular enzyme release in the culture medium is a good in vitro test for quantification of specific tubular damage.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans