Molecular weights of Cucurbita sieve tube proteins

Summary By means of SDS-polyacrylamide-gel electrophoresis, molecular weights of 15000, 28000, 59000, 116000 and 220000 were determined for the main sieve tube proteins from Cucurbita maxima.     

A diurnal component to the variation in sieve tube amino acid content in wheat

We have used high-sensitivity capillary electrophoresis coupled to a laser-induced fluorescence detection method to quantify 16 amino acids in wheat (Triticum aestivum) sieve tube (ST) samples as small as 2 nL collected by severing the stylets of feeding aphids. The sensitivity of the method was sufficient to determine a quantitative amino acid profile of individual STs without the need to bulk samples to produce larger volumes for analysis. This allowed the observation of the full range of variation that exists in individual STs. Some of the total concentrations of amino acids recorded are higher than those reported previously. The results obtained show variation in the concentrations of phenylalanine (Phe), histidine/valine (His/Val), leucine/isoleucine (Leu/Ile), arginine, asparagine, glutamine, tyrosine (Tyr), and lysine (Lys) across the ST samples. These could not be explained by plant-to-plant variation. Statistical analyses revealed five analytes (Tyr, Lys, Phe, His/Val, and Leu/Ile) that showed striking covariation in their concentrations across ST samples. A regression analysis revealed a significant relationship between the concentrations of Tyr, Lys, Phe, Leu/Ile, His/Val, asparagine, arginine, and proline and the time of collection of ST samples, with these amino acids increasing in concentration during the afternoon. This increase was confirmed to occur in individual STs by analyzing samples obtained from stylet bundles exuding for many hours. Finally, an apparent relationship between the exudation rate of ST sap and its total amino acid concentration was observed: samples containing higher total amino acid concentrations were observed to exude from the severed stylet bundles more slowly.     

On sieve tube function

Summary Several theories of phloem transport are currently being considered in various laboratories on the basis of recent evidence. Proponents of the activated diffusion or protoplasmic theories claim support in the disclosure of fibrils, longitudinally arranged, in the connecting strands passing across sieve plates, and in the close connection between respiratory activity and transport. Those favoring a surface migration theory claim support in the demonstrated systems of sieve element lamellae, along whose surfaces one could imagine solute transport to occur. Proponents of the pressure flow theory point to results of exudation studies, tracer investigations, and to histochemical evidence indicating that sieve elements are relatively inactive, metabolically, as well as to the suggestion that perhaps the connecting strands are more open (tubular) than they have been considered to be up to now.Callose formation is stimulated by turgor changes, promoted by foreign chemicals, viruses, and, in the sieve element, by a relatively alkaline pH, a high sucrose concentration, and doubtless by the unique unbalanced character of sieve sap composition. The function of callose in older or wounded elements appears to be a constricting or plugging action, but its function in young mature elements is essentially obscure.Recent evidence augments the view that sieve elements display an extraordinary sensitivity towards experimental manipulation.     

Lignified sieve elements in the wheat leaf

Summary Sieve elements with thick, lignified walls are identified in the longitudinal bundles of the wheat leaf and appear to be functional. They are also present in several festucoid grasses but appear to be absent from the panicoid grasses examined.     

Measurement of the sieve tube membrane potential

A procedure is described for the measurement of the sieve tube membrane potential in the phloem of bark strips from Salix exigua Nutt. Measurements were made by inserting a measuring microelectrode into sap exuding from severed stylets of the willow aphid, Tuberolachnus salignus. Data taken from 20 bark strips gave an average potential of −155 ± 9 millivolts. Evidence is presented for an electrogenic component of the sieve tube membrane potential. The occurrence of a saturable sucrose-induced membrane depolarization is consistent with the concept of sugar accumulation by a sucrose/H⁺ co-transport mechanism.     

Calcium-binding protein in sieve tube exudate

A calcium-binding macromolecule, with an estimated molecular weight greater than 100,000, was detected in phloem exudate from Cucurbita maxima and related species. The macromolecule was a component of sieve tube sap, rather than a contaminant leached from cell walls or cut parenchyma cells during exudate collection. The protein nature of this macromolecule was deduced from its size, lability, susceptibility to proteolytic digestion, and by the dependence of calcium-binding activity on thiol-protecting agents. This protein is distinct from the major proteins of exudate and does not appear to be related to calmodulin.Abbreviations SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - CBP calcium-binding protein     

Turnover of proteins in the extreme thermophileThermus flavus

Thermus flavus contained a limited fraction of short-lived protein when growing in a complex medium; the residual proteins were stable. When incubated in buffer, the residual protein fraction was also degraded. The extent of the short-lived protein fractions was increased by increasing the temperature.     

WCS120 protein family and proteins soluble upon boiling in cold-acclimated winter wheat

The amount of proteins soluble upon boiling (especially WCS120 proteins) and the ability to develop frost tolerance (FT) after cold acclimation was studied in two frost-tolerant winter wheat cultivars, Mironovskaya 808 and Bezostaya 1. Protein gel blot analysis, mass spectrometry (MS) and image analysis of two-dimensional gel electrophoresis (2-DE) gels were used to identify and/or quantify the differences in protein patterns before (non-acclimated, NA) and after 3 weeks of cold acclimation (CA) of the wheats, when FT increased from -4 degrees C (lethal temperature (LT(50)), for both cultivars) to -18.6 degrees C in Bezostaya 1 and -20.8 degrees C in Mironovskaya 808. Only WCS120 protein was visible in NA leaves while all five WCS120 proteins were induced in the CA leaves. Mironovskaya 808 had higher accumulation of three members of WCS120 proteins (WCS120, WCS66 and WCS40) than Bezostaya 1. MS analysis of total sample of proteins soluble upon boiling showed seven COR proteins in the CA samples and only three COR proteins in the NA samples of cultivar Mironovskaya 808 (MIR). In conclusion, the level of the accumulation of WCS120, WCS66 and WCS40 distinguished our two frost-tolerant winter wheat cultivars. Moreover, the differences of CA and NA samples of the MIR were shown by liquid chromatography (LC)-tandem mass spectrometry (MS/MS).     

Some observations on the role of ATP in sieve tube translocation

Summary Using the aphid stylet technique ¹⁴C ATP was shown to be readily taken up into the sieve elements of willow. At the same time this compound was found to be metabolised during uptake resulting in labelled ADP and AMP appearing in the stylet exudate. Longitudinal movement of labelled ATP was also found to occur.Measurement of the levels of ATP and ADP in stylet exudate showed that both were present in high concentrations. The ratio ATP/ADP varied between 2.0 and 5.3.The effect of certain inhibitors of oxidative phosphorylation (oligomycin and DNP) and glycolysis (fluoride) on the rate of stylet exudation was studied. All three inhibitors caused a cessation of exudation but this did not occur until several hours after inhibitor application. Oligomycin and DNP had no effect on the concentration of ATP in the sap. Fluoride however, appeared in some cases to reduce the ATP concentration to a low level an hour or more before exudation finally stopped.Incorporation of ³²Pi into organic phosphate esters present in stylet exudate was found to occur within 15 minutes of the application of the tracer to a bark strip. Labelling of organic phosphates also took place, at a slower rate, when ³²P inorganic phosphate was incubated with stylet exudate.     

The identification of foam-forming soluble proteins from wheat (Triticum aestivum) dough

Proteomic methods have been used to identify foam-forming soluble proteins from dough that may play an important role in stabilising gas bubbles in dough, and hence influence the crumb structure of bread. Proteins from a soluble fraction of dough (dough liquor) or dough liquor foam have been separated by two-dimensional gel electrophoresis, and 42 identified using a combination of matrix-assisted laser desorption/ionization-time of flight and quadrupole-time of flight analyses. Major polypeptide components included beta-amylase, tritin and serpins, with members of the alpha-amylase/trypsin inhibitor family being particularly abundant. Neither prolamin seed storage proteins nor the surface-active protein puroindoline were found. Commonly used dough ingredients (NaCl, Na L-ascorbate) had only a minor effect on the 2-DE protein profiles of dough liquor, of which one of the more significant was the loss of 9 kDa nonspecific lipid transfer protein. Many proteins were lost in dough liquor foam, particularly tritin, whilst a number of alpha-amylase inhibitors were more dominant, suggesting that these are amongst the most strongly surface-active proteins in dough liquor. Such proteins may play a role determining the ability of the aqueous phase of doughs, as represented by dough liquor, to form an elastic interface lining the bubbles, and hence maintain their integrity during dough proving.     

Chromosome localization of genes for soluble proteins in hexaploid wheat (triticum aestivum L.)

An electrophoretic spectra of proteins, extracted with tris-HCI buffer, pH 8.3 are studied. The ditelosomic lines of the Chinese Spring common wheat cultivar are analysed by the chromosomes of the B genome and of the ditelosomic lines of the same cultivar by first and third chromosomes of the D genome. It is found that structural genes for the synthesis of components Nos. 7, 8, 9 and 10 are localized in 1BL, 2BS, 4BS and 5B chromosomes respectively. The genetic control of the component No. 3 is realized by genes, localized in 1BL and 3D chromosomes, while for component No. 2, in the 3D chromosome.     

Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

Soluble and filamentous proteins in Arabidopsis sieve elements

Phloem sieve elements are highly differentiated cells involved in the long-distance transport of photoassimilates. These cells contain both aggregated phloem-proteins (P-proteins) and soluble proteins, which are also translocated by mass flow. We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to carry out a proteomic survey of the phloem exudate of Arabidopsis thaliana, collected by the ethylenediaminetetraacetic acid (EDTA)-facilitated method. We identified 287 proteins, a large proportion of which were enzymes involved in the metabolic precursor generation and amino acid synthesis, suggesting that sieve tubes display high levels of metabolic activity. RNA-binding proteins, defence proteins and lectins were also found. No putative P-proteins were detected in the EDTA-exudate fraction, indicating a lack of long-distance translocation of such proteins in Arabidopsis. In parallel, we investigated the organization of P-proteins, by high-resolution transmission electron microscopy, and the localization of the phloem lectin PP2, a putative P-protein component, by immunolocalization with antibodies against PP2-A1. Transmission electron microscopy observations of P-proteins revealed bundles of filaments resembling strings of beads. PP2-A1 was found weakly associated with these structures in the sieve elements and bound to plastids. These observations suggest that PP2-A1 is anchored to P-proteins and organelles rather than being a structural component of P-proteins.     

Potassium permanganate fixative and the electron microscopy of sieve tube contents

Summary Sieve tube slime is probably fibrillar as suggested by other workers. The granular material seen in electron micrographs of sieve tubes fixed with potassium permanganate is mostly a precipitate produced by the reaction of the permanganate with the sieve tube contents, particularly with sucrose, and with reagents used in the fixing, dehydrating and embedding process. Potassium permanganate is not therefore a good fixative for the electron microscopy of sieve tube contents. Neither is it suitable for the study of fine structure in vacuoles or vesicles containing precipitable solutes in other kinds of cells nor for examining the fine structure of their ground cytoplasm.     

Fine structure and development of phloem sieve tube content

Summary The content of the mature sieve tube ofAcer phloem consists of a network of 80–100 å fibrils. The development of these fibrils from the fibrous masses found in the developing sieve tube is traced and their possible functional significance in translocation discussed.