ADP-Glucose Transport by the Chloroplast Adenylate Translocator Is Linked to Starch Biosynthesis

In organello starch biosynthesis was studied using intact chloroplasts isolated from spinach leaves (Spinacia oleracea). Immunoblot analysis using a specific antiserum against the mitochondrial adenylate (ADP/ATP) translocator of Neurospora crassa shows the presence of an adenylate translocator protein in the chloroplast envelope membranes, similar to that existing in mitochondria and amyloplasts from cultured cells of sycamore (Acer pseudoplatanus). The double silicone oil layer-filtering centrifugation technique was employed to study the kinetic properties of adenylate transport in the purified chloroplasts; ATP, ADP, AMP, and most importantly ADP-Glc were shown to be recognized by the adenylate translocator. Similar to the situation with sycamore amyloplasts, only ATP and ADP-Glc uptake was inhibited by carboxyatractyloside, an inhibitor of the mitochondrial adenylate translocator. Evidence is presented to show that the ADP-Glc transported into the chloroplast stroma is utilized for starch synthesis catalyzed by starch synthase (ADP-Glc:1,4-α-d-glucan 4-α-d-glucosyltransferase). The high activity of sucrose synthase producing ADP-Glc observed in the extrachloroplastic fractions suggests that starch biosynthesis in chloroplasts may be coupled with the direct import of ADP-Glc from the cytosol.     

Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F₁ progeny. Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHA 1, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and β-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F₁ generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F₁ progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.     

Lactate Dehydrogenase in Oryza sativa L. Seedlings and Roots: Identification and Partial Characterization

A lactate dehydrogenase activity is present in rice (Oryza sativa L.) seedlings and roots. Under aerobic conditions, lactate dehydrogenase activity is barely detectable in rice seedlings and is very low in rice roots. In 30 day old roots, the activity is increased two to three times by an anoxic or hypoxic treatment and can be detected on immunoblots by an antiserum raised against barley lactate dehydrogenase. The activity present in aerobic seedlings was partially purified. The native enzyme has a molecular mass of 160 kilodaltons, and is a tetramer of 2 subunit (38 and 39 kilodaltons) randomly associated. Studies of substrate specificity, native gel electrophoresis, and immunoblot analysis indicate that the partially purified enzyme is a typical lactate dehydrogenase. However, no increase of lactate dehydrogenase activity or protein was observed in seedlings transferred to anoxia.     

Control of Nitrogenase mRNA Levels by Products of Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120

Nitrate inhibited nitrogenase synthesis and heterocyst development in the cyanobacterium Anabaena sp. strain PCC 7120. Inhibition of dinitrogen fixation by nitrate did not take place, however, in nitrate reductase-deficient derivatives of this strain. Hybridization of total RNA isolated from cells grown on different nitrogen sources with an internal fragment of the nifD gene showed that regulation of nitrogenase activity by nitrate is exerted through a negative control of the nitrogenase mRNA levels.     

Differential expression of the two Arabidopsis nitrate reductase genes

The differential regulation of the two nitrate reductase (NR, EC 1.6.6.1) genes of Arabidopsis thaliana L. Heynh was examined. cDNAs corresponding to each of the NR genes (NR1 and NR2) were used to measure changes in the steady-state levels of NR mRNA in response to nitrate, light, circadian rhythm, and tissue specificity. Although nitrate-induction kinetics of the two genes are very similar, NR1 is expressed in the absence of nitrate at a higher basal level than NR2. Nitrate induction is transient both in the roots and leaves, however the kinetics are different: the induction and decline in the roots precede that in the leaves. Light induces the expression of each of the genes with significantly different kinetics: NR2 reached saturation more rapidly than did NR1. Both genes showed similar diurnal patterns of circadian rhythm, with NR2 mRNA accumulating earlier in the morning.     

In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum

Reversible seryl-phosphorylation contributes to the light/dark regulation of C₄-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malate-sensitivity of the target enzyme has been recently identified as Ser-15 in ³²P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [³²P]phosphopeptides were isolated and purified from in vivo ³²P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more ³²P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be ³²P-phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C₄ PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation-status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C₄-photosynthesis enzyme in vivo.     

Short-term effects of carbon dioxide on carnation callus cell respiration

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O₂ uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO₂ in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO₂ again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO₂/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO₂/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO₂/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO₂) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.     

Fructosyl Transfer between 1-Kestose and Sucrose in Wheat Leaves

The labeling pattern of the sugar moieties of 1-kestose after in vivo pulse labeling with ¹⁴CO₂ was not the same as that after in vitro labeling with ¹⁴C-sucrose. The two fructosyl residues of 1-kestose had similar specific radioactivities after in vitro synthesis, but after in vivo radiolabeling the specific radioactivity of the terminal fructosyl moiety was significantly less than the internal fructosyl moiety. Evidence is presented that the uneven specific radioactivity of in vivo radiolabeling results from enzymatic transfer of terminal fructosyl residue from 1-kestose to sucrose.     

Absorption of phosphorus from the spermatophore through the cuticle of the bursa copulatrix of the butterfly, Calpodes ethlius

In lepidoptera the spermatophore is deposited in a bursa copulatrix, which is lined by a cuticle through which solubilized components of the spermatophore must be absorbed by the underlying epithelium. This study follows the route taken by the phosphorus which, with calcium, forms a significant portion of the spermatophore of Calpodes ethlius in the form of homogeneous, amorphous concretions. Lead nitrate was used in the primary fixative to precipitate the phosphorus in order to follow the pathway taken by it through the cuticle and bursal epithelium. The epicuticle-free pits, which cover the bursal cuticle, are identified as the points of entry, and the epicuticular filaments as major pathways for transport of phosphorus and presumably soluble molecules. Lead phosphate deposits also occurred in the epithelium extracellularly between the apical microvilli, lateral membranes and basal folds, in calcium granules and in large compound structures which appear to be made up, at least in part, of calcium granules. The confinement of the phosphorus to extracellular compartments of the epithelium suggests that its function is to be found elsewhere.     

Fine structure of the spermatozoon of the strepsipteran Xenos moutoni

Spermatozoa of Xenos moutoni De Buysson belonging to the order Strepsiptera (Insecta) were examined by electron microscopy. The spermatozoon was seen to have an elongated head and a tail containing a 9+9+2 axoneme and two mitochondrial derivatives of equal size. The pear-shaped acrosome is characterised by a mono-layered structure and terminates anteriorly forming two pyramidal evaginations. The nucleus exhibits an external portion of dense chromatin and an internal one of uncondensed material. The latter occupies a central position at the base and becomes progressively peripheral at the apex. The tail is long and in its final portion the axoneme loses its elements progressively. These results have been compared with the ultrastructure of the spermatozoa of Coleoptera which have been considered as a sister group of Strepsiptera.