Biochemical Evidence for the Role of the Waxy Protein from Pea (Pisum sativum L.) as a Granule-Bound Starch Synthase.

Proteins were solubilized from starch extracted from developing pea (Pisum sativum L.) embryos and chromatography of these proteins on a Mono-Q column separated two peaks of starch synthase activity. The major activity peak comprised more than 80% of the total activity. This fraction contained only the Waxy protein, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate followed by staining for proteins or by immunoblot. A 77-kD polypeptide associated with the starch granules and presumed by others to be a starch synthase could not be detected in any of the active fractions. The native molecular weight of the solubilized starch synthase was 59,600 [plus or minus] 1700 as determined by sucrose density gradient. It is concluded that in pea seeds the Waxy protein and the starch synthase bound to the granule are the same protein.

Changes in Net O(2) Exchange Induced by Inorganic Nitrogen in the Blue-Green Alga Anacystis nidulans.

The response of net O(2) exchange to light intensity by intact Anacystis nidulans cells in the presence of saturating NaHCO(3) concentrations followed a curve with an inflection near the light-compensation point. Addition of either KNO(3) or NH(4)Cl stimulated O(2) uptake in the dark and at light intensities below the light-compensation point. This resulted in steeper slopes of the curve calculated below and above the light-compensation point. At O(2) concentrations limiting dark respiration, addition of inorganic nitrogen had no effect on either dark respiration or O(2) exchange in the light. The apparent changes in photosynthetic yield observed under normal O(2) concentration disappeared when respiration was limited by O(2) availability, indicating that the effects of inorganic nitrogen on O(2) exchange at low light intensities are due to stimulation of respiration rather than to increases in photosynthetic yield.

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12.

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans.

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.

Carbon Dioxide-Induced Oscillations in Fluorescence and Photosynthesis: Role of Thylakoid Membrane Energization in Regulation of Photosystem II Activity.

The response of CO(2) fixation to a sudden increase in ambient CO(2) concentration has been investigated in intact leaf tissue from spinach (Spinacia oleracea) using a dual channel infrared gas analyzer. Simultaneous with these measurements, changes in fluorescence emission associated with a weak, modulated measuring beam were recorded. Application of brief (2-3 seconds) dark intervals enabled estimation of the dark fluorescence level (F(o)) under both steady state and transient conditions. The degree of suppression of F(o) level fluorescence in the light was strongly correlated with nonphotochemical quenching under all conditions. During CO(2)-induced oscillations in photosynthesis under 2% O(2) the changes in nonphotochemical quenching anticipate changes in the rate of uptake of CO(2). At such low levels of O(2) and constant illumination, changes in the relative quantum efficiency of open photosystem II units were estimated as the ratio of the rate of CO(2) uptake and the photochemical quenching coefficient. Under the same conditions the relative quantum efficiency of photosystem II was found to vary inversely with the degree of nonphotochemical quenching. The relationship between changes in the rate of CO(2) uptake: photochemical quenching coefficient and nonphotochemical quenching was altered somewhat when the same experiment was conducted under 20% O(2). The results suggest that electron transport coupled to reduction of O(2) occurs to varying degrees with time during oscillations, especially when ambient O(2) concentrations are high.

Relationship between Steady-State Fluorescence Yield and Photosynthetic Efficiency in Spinach Leaf Tissue.

The relationship between steady-state photosynthetic efficiency, as moles CO(2) per mole of incident visible photons under 2% O(2), and chlorophyll fluorescence quenching has been investigated in intact leaf tissue of Spinacia oleracia. Fluorescence yield was measured using a pulse amplitude modulation technique that permitted rapid and sensitive resolution and quantitation of photochemical and nonphotochemical quenching coefficients. A highly linear relationship was observed between photosynthetic efficiency and the ratio of photochemical:nonphotochemical quenching coefficients for values of the latter less than 1.6. This relationship applied whether irradiance or CO(2) concentration was varied. The observed relationships between photochemical yield and fluorescence yield were compatible with the photosystem II model proposed by Butler and Kitajima (1975 Biochim Biophys Acta 376: 116-125). The results are discussed with respect to the proposed role of nonphotochemical quenching in regulating radiant energy utilization and also the applicability of fluorescence measurements as a means of estimation of the rate of photosynthetic electron transport.

Sudden changes in the rate of photosynthetic oxygen evolution and chlorophyll fluorescence in intact isolated chloroplasts: the role of orthophosphate.

Simultaneous "ripples" (sudden changes in rate) in CO2 dependent O2 evolution and associated chlorophyll a fluorescence were followed in isolated, largely intact, spinach chloroplasts. These ripples could only be observed under conditions in which the supply of inorganic phosphate was limiting. This limitation was achieved either by 1) omission of phosphate in the assay medium, 2) use of inhibitors of the phosphate translocator, or 3) the addition of triose phosphate, a competitive inhibitor of Pi for the same translocator.The possible relation of these ripples to the dampening oscillations that can be observed in leaves, leaf pieces, isolated cells and protoplasts, is discussed.

The relationship between light scattering and chlorophyll a fluorescence during oscillations in photosynthetic carbon assimilation.

Light scattering, which can be taken as an indicator of the transthylakoid proton gradient, and the 518-nm rise, which can be regarded as a measure of the transthylakoid membrane potential, have been followed during oscillations in chlorophyll a fluorescence, which are known to be associated with corresponding changes in photosynthetic carbon assimilation. Both components oscillated in a manner which was broadly reciprocal to chlorophyll a fluorescence. However, there was a phase shift such that the light-scattering change usually anticipated fluorescence and often also the 518-nm shift. It is concluded that the proton motive force rises and falls slightly in advance of rises and falls in carbon assimilation. The relationship of these changes to a possible underlying mechanism is discussed.

Chlorophyll a fluorescence and light-scattering kinetics displayed by leaves during induction of photosynthesis.

Light-scattering, which can be taken as an indicator of the transthylakoid proton-gradient, and chlorophyll a fluorescence, have been followed simultaneously during re-illumination of spinach leaves at different energy fluence rates and carbon dioxide concentrations. The slow fluorescence transient ("M peak"), which has been associated with photosynthetic induction, was observed in air only at the lower fluence rates used. Data are presented that indicate that M peaks in chlorophyll fluorescence kinetics can only be observed if there is also a simultaneous transient in light-scattering and that these transients are observed when the dark period is relatively long, fluence rate relatively low, and CO2 concentration relatively high.The results are discussed in relation to the varying demands on ATP by carbon assimilation during induction of photosynthesis at different carbon dioxide concentrations and the manner in which these variations influence the quenching of chlorophyll a fluorescence.

Simultaneous measurement of oscillations in oxygen evolution and chlorophyll a fluorescence in leaf pieces.

In spinach (Spinacia oleracea) and barley (Hordeum vulgare) leaves, chlorophyll a fluorescence and O(2) evolution have been measured simultaneously following re-illumination after a dark interval or when steady state photosynthesis has been perturbed by changes in the gas phase. In high CO(2) concentrations, both O(2) and fluorescence can display marked dampening oscillations that are antiparallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O(2) by about 10 to 15 seconds). Infrared gas analysis measurements showed that CO(2) uptake behaved like O(2) evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence. In the steady state, oscillations were initiated by increases in CO(2) or by increases or decreases in O(2). Oscillations in O(2) or CO(2) did not occur without associated oscillations in fluorescence and the latter were a sensitive indicator of the former. The relationship between such oscillations in photosynthetic carbon assimilation and chlorophyl a fluorescence is discussed in the context of the effect of ATP or NADPH consumption on known quenching mechanisms.