Photosynthetic Decline from High Temperature Stress during Maturation of Wheat : II. Interaction with Source and Sink Processes

High temperature stress reduces grain growth in wheat (Triticum aestivum L.) by altering source activity and sink capacity. The impact of stress on source and sink interactions in two wheat cultivars of differing source thermotolerance was monitored by analysis of chlorophyll fluorescence transients, Fv (variable fluorescence) and PSM (peak, stationary, maximum), of attached flag leaves on intact and decapitated tillers grown at optimum (20°C) and stress (35°C) temperatures after anthesis. The thermotolerant cultivar Waverly had reduced Fv and PS quenching and a large increase of SM during heat stress. The less thermotolerant cultivar, Len, exhibited increased Fv and PS quenching and a small increase of SM. Fluorescence induction was similar in intact and decapitated tillers of Len, indicating diminished sinksource interaction during heat stress. The present results and previous observations of photosynthetic activities indicate that cyclic electron transport and photophosphorylation in flag leaves of the thermotolerant cultivar were stimulated by sink demand (increased SM in intact plants). Reduced grain development in the thermolabile cultivar resulted from limited capacity to support cyclic electron transport and photophosphorylation (slight increase in SM of intact plants and large reduction of Cytochrome f/b₆-mediated electron transport capacity). It was concluded that heat stress injures the photosynthetic apparatus during reproductive growth of wheat and that diminished source activity and sink capacity may be equally important in reducing productivity.     

Reconstitution of pigment-containing complexes from light-harvesting chlorophyll a/b-binding protein overexpressed inEscherichia coli

A gene for a light-harvesting chlorophyll (Chl) a/b-binding protein (LHCP) from pea (Pisum sativum L.) has been cloned in a bacterial expression vector. Bacteria (Escherichia coli) transformed with this construct produced up to 20% of their protein as pLHCP, a derivative of the authentic precursor protein coded for by the pea gene with three amino-terminal amino acids added and-or exchanged, or as a truncated LHCP carrying a short amino-terminal deletion into the mature protein sequence. Following the procedure of Plumley and Schmidt (1987, Proc. Natl. Acad. Sci. USA84, 146–150), all bacteria-produced LHCP derivatives can be reconstituted with acetone extracts from pea thylakoids or with isolated pigments to yield pigment-protein complexes that are stable during partially denaturing polyacrylamide-gel electrophoresis. The spectroscopic properties of these complexes closely resemble those of the light-harvesting complex associated with photosystem II (LHCII) isolated from pea thylakoids. The pigment requirement for the reconstitution is highly specific for the pigments found in native LHCII: Chl a and b as well as at least two out of three xanthophylls are necessary. Varying the Chl a:Chl b ratios in the reconstitution mixtures changes the yields of complex formed but not the Chl a:Chl b ratio in the complex. We conclude that LHCP-pigment assembly in vitro is highly specific and that the complexes formed are structurally similar to LHCII. The N-terminal region of the protein can be varied without affecting complex formation and therefore does not seem to be involved in pigment binding. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Accumulation of a light-harvesting chlorophyll a/b protein in the chloroplast grana lamellae. The lateral migration of the membrane protein precursor is independent of its processing.

The events that follow the import of pLHCPIIb, the apoprotein precursor of the major light-harvesting complex of photosystem II, were studied in intact pea chloroplasts. The distribution of the events of insertion into the membrane, and processing, to yield the mature form (LHCP) between stromal and granal lamellae regions of the thylakoids were followed. pLHCP was preferentially inserted into stromal lamellae (SL) from which it migrated to granal lamellae (GL). Migration occurred before or after processing, suggesting that migration and processing are independent of each other. When migration was slowed down, LHCP accumulated in SL. Prolonged inhibition of migration induced degradation of LHCP that had accumulated in SL, whereas inhibition of processing did not affect the migration of pLHCP into GL. A small difference in electrophoretic mobility was noted between LHCP in SL and in GL. The predominant mature form in SL migrated more slowly than LHCP from GL. When thylakoids were subjected to trypsin, all of the LHCP embedded in SL underwent cleavage, whereas up to 60% of the radioactive LHCP in GL was resistant to the enzyme. The possible implications of the differences in size and in the sensitivity to trypsin of LHCP are discussed.     

Light-harvesting chlorophyll protein (LHCII) drives electron transfer in semiconductor nanocrystals

Type-II quantum dots (QDs) are capable of light-driven charge separation between their core and the shell structures; however, their light absorption is limited in the longer-wavelength range. Biological light-harvesting complex II (LHCII) efficiently absorbs in the blue and red spectral domains. Therefore, hybrid complexes of these two structures may be promising candidates for photovoltaic applications. Previous measurements had shown that LHCII bound to QD can transfer its excitation energy to the latter, as indicated by the fluorescence emissions of LHCII and QD being quenched and sensitized, respectively. In the presence of methyl viologen (MV), both fluorescence emissions are quenched, indicating an additional electron transfer process from QDs to MV. Transient absorption spectroscopy confirmed this notion and showed that electron transfer from QDs to MV is much faster than fluorescence energy transfer between LHCII and QD. The action spectrum of MV reduction by LHCII-QD complexes reflected the LHCII absorption spectrum, showing that light absorbed by LHCII and transferred to QDs increased the efficiency of MV reduction by QDs. Under continuous illumination, at least 28 turnovers were observed for the MV reduction. Presumably, the holes in QD cores were filled by a reducing agent in the reaction solution or by the dihydrolipoic-acid coating of the QDs. The LHCII-QD construct can be viewed as a simple model of a photosystem with the QD component acting as reaction center.     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

Intracellular Localization of Phospholipase D in Leaves and Seedling Tissues of Castor Bean

The intracellular distribution of phospholipase D (PLD; EC 3.1.4.4) in castor bean (Ricinus communis L.) tissues was investigated by subcellular fractionation and by immuno-electron microscopy. Centrifugal fractionation revealed that most PLD in young leaves was soluble, whereas in mature leaves a majority of PLD was associated with microsomal membranes. Further separation of microsomal membranes by a two-phase partitioning system indicated that PLD was associated with both plasma and intracellular membranes. Sucrose gradient separation of intracellular membranes showed PLD present in the endoplasmic reticulum, a submicrosomal band, and in soluble fractions but not in mitochondria and glyoxysomes of postgermination endosperm. Immunocytochemical studies found high gold labeling in vacuoles in young leaves, suggesting that the high level of soluble PLD in young leaves is due to release of PLD from vacuoles during tissue disruption. In addition to the labeling in vacuoles, gold particles were also found in the cytoplasmic matrices and plasma membrane in leaves and in 2-d postgermination seedlings. Collectively, these results show that PLD in castor bean leaf and seedling tissues is localized in the vacuole and is associated with the endoplasmic reticulum and plasma membrane and that the relative distribution between the soluble and membrane compartments changes during castor bean leaf development.     

Further characterization of a high molecular weight glycoprotein antigen from the yeast Saccharomyces cerevisiae

A high molecular weight glycoprotein antigen was isolated by size exclusion chromatography on Sepharose 4B from an extract of the yeast Saccharomyces cerevisiae. The glycoprotein antigen Sc 500 was shown to be identical to the antigen termed gp200 previously isolated (Heelan et al., 1991). The MW of Se 500 was determined to be about 500 kDa by size exclusion chromatography on Superose 6 and 460 kDa ± 20k Da by size-exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). Sc 500 contained 90% mannose and traces of N-acetylglucosamine. The amino acid composition revealed that serine and threonine were the most abundant amino acids of the protein part. By alkaline borohydride treatment some, but not all bonds between protein and carbohydrate were broken. This indicates that the main type of linkage between protein and carbohydrate is O-glycosidic and that a minor type is of N-glycosidic nature. Methylation analysis revealed that the mannose residues were connected by 1 → 2 and 1 → 3 linkages with 1 → 2, 1→ 6 linked branch points.Purified Sc 500 was subjected to a series of chemical and enzymatic modifications followed by studies of antibody binding activity. Treatments with both periodate and alkaline sodium borohydride reduced the human serum IgA, IgG and monoclonal IgM antibody binding activity of Sc 500 whereas trypsin and pronase did not affect its ability to bind these antibodies. The mannosidase Manα1 → 2,3,6Man reduced the IgM binding to Sc 500 while the other enzymes included in this experiment (Manα1→2 Man, Manβ1 →4GlcNAc and PNGase F) had no effect on the antibody binding.     

Synthesis of O-glycan core 3: Characterization of UDP-GlcNAc: GalNAc-R {beta}3-N-acetyl-glucosaminyltransferase activity from colonic mucosal tissues and lack of the activity in human cancer cell lines

UDP-GlcNAc: GalNAc-R ß3-GlcNAc-transferase (core 3ß3-GlcNAc-T, where GlcNAc is N-acetyl-D-glucosamine,GalNAc is N-acetyl-D-galactosamine and T is transferase) isexpressed in a tissue-specific fashion and is high in normalcolonic tissue, but downregulated in colon cancer. To furtherstudy the control of this enzyme, we examined the activity inpig, rat and human colonic tissues, and several human cancercell lines. The enzyme was difficult to solubilize by detergentsand was extremely unstable in the solubilized form. Using syntheticderivatives of the GalNAc-R substrate, we showed that the specificityof the enzyme in normal rat and human colonic mucosa requiresall the substituents of the GalNAc-sugar ring of substratesfor maximal activity. Core 3 ß3-GlcNAc-T was significantlyinfluenced by the structure of the aglycon group. None of theinactive substrate derivatives could inhibit the activity. N-Iodoacetamido-galactosamine     

Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM-3 by molecular dynamics simulations

Molecular dynamics trajectories were calculated separately for each of the two molecules in the asymmetric unit of the crystal structure of the hemoprotein domain of cytochrome P450BM-3. Each simulation was 200 ps in length and included a 10 Å layer of explicit solvent. The simulated time-average structure of each P450BM-3 molecule is closer to its crystal structure than the two molecular dynamics time-averaged structures are to each other. In the crystal structure, molecule 2 has a more accessible substrate binding pocket than molecule 1, and this difference is maintained throughout the simulations presented here. In particular, the substrate docking regions of molecule 1 and molecule 2 diverge in the solution state simulations. The mouth of the substrate binding pocket is significantly more mobile in the simulation of molecule 2 than in the simulation of molecule 1. For molecule 1, the width of the mouth is only slightly larger than its X-ray value of 8.7 Å and undergoes fluctuations of about 1 Å. However, in molecule 2, the mouth of the substrate binding pocket is dramatically more open in the time-average molecular dynamics structure (14.7 Å) than in the X-ray structure (10.9 Å). Furthermore, this region of the protein undergoes large amplitude motions during the trajectory that are not seen in the trajectory of molecule 1, repeatedly opening and closing up to 7 Å. Presumably, the binding of different substrates will induce the mouth region to adopt different conformations from within the wide range of structures that are accessible. © 1995 Wiley-Liss, Inc.