Albert R. Carlier

Reversible and irreversible activation ofPhycomyces blakesleeanus spores

Phycomyces spores do not germinate in a suitable culture medium unless they are activated by one of a range of treatments. Heating the spores for 3 min at 44°C resulted in nearly complete germination in culture medium; however, when such spores were incubated in water, rapid deactivation occurred. Deactivation of spores treated for 3 min at 50°C was much slower and only partial. After reversible activation (44°C) RNA and protein synthesis increased rapidly in culture medium but not in water. Immediately after irreversible activation (50°C) uridine and leucine incorporation was severely reduced but increased rapidly upon further incubation in both water and culture medium. Activation of spores at 44 or 50°C in culture medium had a similar effect on nucleoside triphosphate content: the ATP level was high and did not change markedly after activation; the UTP and GTP content, however, showed a clear-cut increase shortly after activation. Spores incubated in water had a much lower nucleoside triphosphate content but upon irreversible activation (50°C) the pattern of the nucleoside triphosphates was similar to that in culture medium. Reversible activation (44°C) of spores incubated in water yielded only a temporary increase in ATP level. The pattern of respiration was the same for reversibly and irreversibly activated spores in culture medium. During incubation in water, however, irreversibly activated spores had a higher respiration than reversibly activated spores. This suggests that the irreversibility of activation at 50°C was caused by the occurrence of the initial phases of germination, even in water, whereas after reversible activation external carbon sources were required to start germination and to maintain the activated state. Respiration was insensitive to azide in dormant spores but became progressively more sensitive during germination.

Trehalase Activity in Dormant and Activated Spores of Phycomyces blakesleeanus

SummaryHeat treatment of Phycomyces sporangiospores, which breaks dormancy, causes a very rapid 10- to 15fold increase in trehalase activity; soon after the heat shock the enzyme activity decays. This phenomenon can be repeated several times by repeating the heat shocks. Prolonging the heat treatment over the minimum required time delays the decay of enzyme activity. Cycloheximide does not prevent the rise in enzyme activity. It is suggested that heat treatment converts temporarily an inactive form of trehalase into an active one. Optimal enzyme activity is obtained at pH 7.5 and the enzyme requires metal ions for maximal activity. The possible role of trehalase in the spore-activation process is discussed.

Preformed and newly synthesized messenger RNA in germinating wheat embryos

In 6 h germinated wheat (Triticum aestivum L. cv. Cama) embryos, more than half of the messenger RNAs are actively involved in translation. Neither preformed nor newly synthesized poly A+-RNA is translated preferentially. Germination in the presence of cordycepin showed that the half-life of the templates is about 2 h and that the newly synthesized messengers are essential to support protein synthesis in the embryo from the first hours of germination. Most of the messenger RNAs in 6 h germinated embryos are newly synthesized. The polypeptides coded for by either the endogenous messenger ribonucleoproteins or purified poly A+-RNA from both dry and germinated embryos are qualitatively identical; minor quantitative differences can however be observed.

A genetic basis for the origin of six different isolectins in hexaploid wheat.

Wheat (Triticum aestivum) germ agglutinin represents a complex mixture of multiple isolectin forms. Upon ion exchange chromatography at pH 3.8, three isolectins can be separated, each of which is composed of two identical subunits. At pH 5.0, however, three additional isolectins can be distinguished, which are built up of two different subunits (heteromeric lectins). Evidence is presented that these heterodimers are normal constituents of the wheat embryo cells. Analyses of the isolectin patterns in extracts from Triticum monococcum, Triticum turgidum dicoccum and Triticum aestivum, provide evidence that each genome, either in simple or complex (polyploid) genomes, directs the synthesis of a single lectin subunit species. In addition, a comparison of the isolectin pattern in these wheat species of increasing ploidy level, made it possible to determine unequivocally the genome by which the individual lectin subunits in polyploid species are coded for. The possible use of lectins in studies on the origin of individual genoms in polyploid species is discussed.

A lectin from Bryonia dioica root stocks.

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.

Lectin synthesis in developing and germinating wheat and rye embryos

Wheat (Triticum aestivum L.) and rye (Secale cereale L.) lectins are specifically synthesized during seed formation. They accumulate exponentially in the primary axes in a period coinciding with the development of this complex organ. Since the specific lectin content also increases dramatically, there is apparently an outburst of lectin synthesis during the development of the primary axes. Germinating embryos also synthesize some lectin. The fortunate availability of a highly specific procedure for the isolation of cereal lectins enabled us to follow the kinetics of their synthesis during early germination. Stored mRNAs appear to be involved in this residual lectin synthesis.

Occurrence and immunological relationships of lectins in gramineous species

A survey of the occurrence of lectins in seeds from more than 100 grass species showed that all species belonging to the Triticeae tribe and the genera Brachypodium and Oryza contain lectins. All these lectins have the same sugar-binding specificity and are related to wheat-germ agglutinin, but to different degrees. Lectins from Triticeae species are immunologically indistinguishable from wheat lectin, whereas Brachypodium and rice lectins are only immunologically related to the wheat lectin. Attempts to detect lectin-deficient lines or varieties in wild and cultivated species of the three lectin-containing groups were unsuccessful. The possible use of lectins as a chemotaxonomic tool is discussed.

The rye embryo system as an alternative to the wheat system for protein synthesis in vitro.

Isolated rye embryos are a readily available source for the preparation of very active, cell-free, protein-synthesizing systems. Incorporation levels up to 2000 pmol leucine per 50 mul assay are routinely obtained at saturating TMV (Tobacco mosaic virus) RNA concentrations; at limiting messenger RNA concentrations the incorporation exceeds 1000 leucine molecules per TMV RNA molecule. The characteristics of this cell-free system for the translation of TMV RNA are identical with those of a similarly prepared wheat germ system. The major advantage of the rye embryo system is its high reliability as compared to the unpredictable wheat germ system. Sucrose gradient analysis of the reaction mixture during the incubation shows an extensive polysome formation with TMV RNA and demonstrates efficient polypeptide chain release.

Synthesis and proteolytic activation of chitin synthetase in Phycomyces blakesleeanus Burgeff

The chitin synthetase of Phycomyces blakesleeanus mycelium is a particulate enzyme sedimenting mostly at 1000xg. The activity in crude extracts or cellular fractions can be increased more than tenfold by mild trypsin treatment. Plotting the reaction velocity versus UDP-N-acetylglucosamine concentration yields a sigmoidal curve. N-acetylglucosamine, which greatly stimulates the enzyme, changes the kinetics to an almost normal hyperbolic relationship.The enzyme is nearly absent in dormant spores and is synthesized “de novo” in germinating spores (from 4 h germination on). Trypsin treatment of extracts from germinating spores to assay the synthesis of the proenzyme did not reveal an earlier synthesis of the zymogen, which therefore might have some activity of its own.

Messenger ribonucleoprotein particles in dry wheat and rye embryos

Extracts prepared from dry wheat (Triticum aestivum L.; var. Cama) and rye (Secale cereale L.; var. Celestijner) embryos exhibit relatively high endogenous messenger activities in an in vitro protein synthesizing system: up to 400 pmol leucine are incorporated per 50 μl reaction mixture; the messenger/ribosome ratio is estimated at about 0.03. Sucrose gradient analysis of the incubation mixture during cellfree protein synthesis shows a progressive in vitro polysome formation with the native endogenous mRNPs. The translation of these messengers, although dependent on initiation, follows linear kinetics. The native mRNP particles can be separated on sucrose gradients in 6 distinct size classes with sedimentation values varying from 25 S to 104 S. Their size is strongly reduced after deproteinization so that the naked mRNAs sediment in a range between 8 S and 35 S. The molecular weights of the proteins synthesized under direction of the native RNA particles range from 12,000 to 70,000 as estimated by polyacrylamide gel electrophoresis.