Cell-type specific,coordinate expression of two ADP-glucose pyrophosphorylase genes in relation to starch biosynthesis during seed development of Vicia faba L.

Several cDNA clones encoding two different ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) polypeptides denoted VfAGPC and VfAGPP were isolated from a cotyledonary library of Vicia faba L. Both sequences are closely related to AGPase small-subunit sequences from other plants. Whereas mRNA levels of VfAGPP were equally high in developing cotyledons and leaves, the mRNA of VfAGPC was present in considerable amounts only in cotyledons. During development of cotyledons, both mRNAs accumulated until the beginning of the desiccation phase and disappeared afterwards. The increase of AGPase activity in cotyledons during the phase of storage-product synthesis was closely followed by the accumulation of starch. The AGPase activity in crude extracts of cotyledons was insensitive to 3-phosphoglycerate whereas the activity from leaves could be activated more than five-fold. Inorganic phosphate inhibited the enzyme from both tissues but was slightly more effective on the leaf enzyme. There was a correlation at the cellular level between the distribution of VfAGPP and VfAGPC mRNAs and the accumulation of starch, as studied by in-situ hybridisation and by histochemical staining in parallel tissue sections of developing seeds, respectively. During the early phase of seed development (12–15 days after fertilization) VfAGPase mRNA and accumulation of starch were detected transiently in the hypodermal, chlorenchymal and outer parenchymal cell layers of the seed coat but not in the embryo. At 25 days after fertilization both synthesis of VfAGPase mRNA and biosynthesis of starch had started in parenchyma cells of the inner adaxial zone of the cotyledons. During later stages, the expression of VfAGPase and synthesis of starch extended over most of the cotyledons but were absent from peripheral cells of the abaxial zone, provascular and procalyptral cells.Abbreviations AGPase ADP-glucose pyrophosphorylase - DAF days after fertilization - Glc1P glucose-1-phosphate - 3-PGA 3-phosphoglycerate - VfAGPC AGPase subunit of Vicia faba mainly expressed in cotyledons - VfAGPP AGPase subunit of Vicia faba mainly expressed in leaves and cotyledons - pVfAGPC, pVfAGPP plasmids containing VfAGPC and VfAGPP, respectively This work was supported by the Bundesministerium für Forschung und Technologie BCT 0389, Molekular- und Zellbiologie von höheren Pflanzen und Pilzen. U.W acknowledges additional support by the Fonds der chemischen Industrie. We thank Elsa Fessel for excellent technical assistance.     

Ferredoxin-Dependent and Antimycin A-Sensitive Reduction of Cytochrome b-559 by Far-Red Light in Maize Thylakoids; Participation of a Menadiol-Reducible Cytochrome b-559 in Cyclic Electron Flow

Thylakoids from mesophyll cells of maize showed a high rateof the ferredoxin (Fd)-dependent and antimycin A (AntiA)-sensitivecyclic electron flow as determined by the quenching of 9-aminoacridinefluorescence which indicates the formation of     

Bioenergetics of Carbon Assimilation in Intact Chloroplasts: Coupling of Proton to Electron Transport at the Ratio H+/e=3 Is Incompatible with H+/ATP=3 in ATP Synthesis

During a transition from aerobic to largely anaerobic conditionslight-saturated carbon assimilation of intact chloroplasts wasnot decreased although both the transthylakoid proton gradientand ATP levels declined. After a dark period under anaerobiosis,illumination failed to initiate carbon assimilation. ATP increasedonly transiently in the light and then returned to the darklevel. Under such conditions, the addition of electron acceptorssuch as oxygen, oxalacetate or nitrite resulted in the increaseof ATP levels and carbon assimilation was initiated. Assimilationcontinued under anaerobiosis in the presence of reduced protongradients and reduced ATP levels after electron acceptors addedin addition to bicarbonate were reduced. Cyclic electron transport was inhibited when anaerobiosis didnot permit linear electron transport. It was induced in thissituation by micromolar concentrations of oxygen or when, underanaerobiosis, DCMU decreased PSII activity. Oxygen inhibitedcyclic electron transport by draining electrons from the cyclicpathway only when electron donation from PSII was weak. Theobservations give evidence of the delicate redox balance requiredfor cyclic electron transport. Since H⁺/e=3 in linear electron transport, the observationsof effective carbon reduction under a decreased transthylakoidproton gradient and decreased levels of ATP are incompatiblewith H⁺/ATP=2 or 3. They are compatible with H⁺/ATP=4. (Received May 1, 1995; Accepted October 3, 1995)     

Suppression of Quantum Yield of Photosystem II by Hyperosmotic Stress in Chlamydomonas reinhardtii

Addition of ethylene glycol (EG) or NaCl to cells of Chlamydomonasreinhardtii induced transient non-photochemical quenching ofChl fluorescence correlated with the inhibition of photosyntheticoxygen evolution. The induction of the quenching and subsequentrecovery proceeded not only in the light but also in the dark.The quenching was almost unaffected by the protonophore nigericin,suggesting the involvement of a type of non-photochemical quenchingattributable to a state 2 transition. Higher concentrationsof EG or NaCl caused a delay of the recovery of the maximumfluorescence yield (Fm'). Dark reduction rate of P700⁺ afterthe application of a flash light in the presence of DCMU wasenhanced by the hyperosmotic shock, suggesting a stimulatedreduction of the intersystem electron carriers. It is proposedthat the osmotic stress stimulates electron donation from stromalcomponents via the NAD(P)H dehydrogenase, which results in thereduction of the intersystem chain and triggering of a state2 transition leading to stimulated cyclic PSI activity. (Received May 16, 1995; Accepted July 26, 1995)     

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3-untranslated regions (3-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3 non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event ocurring in this intron.     

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Mixed-ligand complexes of technetium XIII. A new and facile synthesis, structure and electrochemical behaviour of trans-[Tc(dppe)2(buNC)2](PF6)· ethanol (dppe = bis(diphenylphosphino)ethane, buNC= tert-butylisocyanide)

The Tc(I) mixed-ligand complex, trans-[Tc(dppe)₂(bu^tNC)₂](PF₆) (dppe=bis(diphenylphosphino)ethane, bu^tNC=tert-butyl-isocyanide) has been prepared from [Tc(tu−S)₆³⁺ (tu-S=thiourea) and a mixture of both ligands. The compound crystallizes triclinic in the space group

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). The technetium atom has a slightly distorted octahedral coordination sphere with the isocyanide ligands in trans-position to each other. By cyclic voltammetry, at a Pt electrode, trans-[Tc(dppe)₂(bu^tNC)₂](PF₆) undergoes a single electron reversible oxidation at E_1/2^ox=0.91 V versus SCE.     

The basic organization of the Plathelminthes

The basic organization of the Plathelminthes is summarized. Special attention is given to epidermal structures, musculature, extracellular matrices, nervous system and sensory structures, digestive system, totipotent stem cells, protonephridia, reproductive system and life cycle. The latest common ancestor of the Plathelminthes lacked any parenchymal cells and tissues; Plathelminthes do not represent Parenchymia. Discussions concerning the relationships of the Plathelminthes with other Metazoa must be based on the characteristics of the plathelminth stem species.     

Specific phosphatidylethanolamine dependence of Serratia marcescens cytotoxin activity

The cytolytic and haemolytic activity of Serratia marcescens is determined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein. In the absence of ShlB, inactive ShlA* remains in the periplasm of Escherichia coli transformed with an shlA-encoding plasmid, which indicates that ShlB converts ShlA* to active ShlA. ShlA* in a periplasmic extract and partially purified ShlA* were activated in vitro by partially purified ShlB. When both proteins were highly purified, ShlA* was only activated by ShlB when phosphatidylethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA* activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysaccharide and various detergents could not substitute for PE. Although radioactively labelled PE was so tightly associated with ShlA that it remained bound to ShlA after heating and SDS–PAGE, it was not covalently linked to ShlA as PE could be removed by thin-layer chromatography with organic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 ± 2.2. Active ShlA was inactivated by treatment with phospholipase A₂, which indicated that PE is also required for ShlA activity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) reversibly complemented ShlA* to active ShlA and was inactivated by phospholipase A₂, which demonstrated that PE binds to the N-terminal portion of ShlA; this region has previously been found to be involved in ShlA secretion and activation. Electrospray mass spectroscopy of ShlA-255 determined a molar mass that corresponded to that of unmodified ShlA-255. An E. coli mutant that synthesized only minute amounts of PE did not secrete ShlA but contained residual cell-bound haemolytic activity. Since PE binds strongly to ShlA* in the absence of ShlB without converting ShlA* to haemolytic ShlA, ShlB presumably imposes a conformation on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic membrane.     

Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates.