Free amino Acid composition of leaf exudates and Phloem sap : a comparative study in oats and barley

Comparisons were made between the free amino acid composition in leaf exudates and that in pure phloem sap, using twin samples taken from a single leaf of two oat (Avena sativa L.) and three barley (Hordeum vulgare L.) varieties. Leaf exudate was collected in a 5 mm EDTA-solution (pH 7.0) from cut leaf blades and phloem sap was obtained through excised aphid (Rhopalosiphum padi L.) stylets. Fluorescent derivatives of amino acids were obtained using 9-fluorenylmethyl chloroformate and were separated by means of high performance liquid chromatography. The total concentration of free amino acids varied considerably in the exudate samples. There was no correlation between the total amino acid content in the exudate samples and that of the corresponding phloem sap samples, but the amino acid composition of the corresponding samples was highly correlated (median R²-value 0.848). There was only limited between-plant variation in phloem sap amino acid composition. Nevertheless, in comparisons involving all samples, many of the amino acids showed significant correlations between their relative amounts in exudate and phloem sap. The results presented here indicate that the exudate technique holds great promise as an interesting alternative to the laborious and time-consuming stylet-cutting technique of obtaining samples for comparative studies of phloem sap.     

A Histological Evaluation of Induced Resistance to Phytophthora infestans (Mont.) de Bary in Potato Leaves

Systemically induced resistance against Phytophthora infestans in the potato cultivar ‘Bintje’ was studied histologically at the light microscopy level on the leaf surface, in the epidermis and in the mesophyll of challenged potato leaves. Systemic disease resistance was induced by a local pre-infection with the same fungus. On the leaf surface of induced plants, the germination of cysts was enhanced. In the epidermis of induced plants, papilla deposition increased and penetration decreased, whereas in the mesophyll of induced plants the spread of hyphae was reduced. It is suggested that the reduction of disease severity in induced plants is the result of the combined action of several successive defence reactions.     

A poly(U) polymerase in tobacco leaves.

A poly(U) polymerizing enzyme has been found in healthy and tobacco mosaic virus-infected tobacco leaves and has been partially purified by affinity chromatography on a gel prepared from agarose with chemically coupled RNA. The enzyme is stimulated by Mn-2+ and dependent on a polynucleotide, preferentially poly(A). The synthesis proceeds optimally at pH 7.6 and 25 degrees C. The enzyme is highly specific for UTP and is inhibited by other ribonucleoside triphosphates. The product was partly sensitive to pancreatic ribonuclease. The synthetic reaction is inhibited in the presence of pyrophosphate but insensitive to 10 mM orthophosphate and high levels of cordycepin, rifampicin and actinomycin D. A molecular weight of about 40,000 has been estimated by sucrose gradient analysis and partition cell ultracentrifugation.     

RNA-synthesizing enzymes in healthy and TMV-infected tobacco leaves. Separation and properties of enzymes catalyzing nucleotide polymerization

A separation procedure, preparation of the extract and a gel filtration on 8% agarose, has been developed for the separation of enzymes catalyzing nucleotide polymerization in healthy and TMV-infected tobacco leaves. The profiles of the gel filtrations were almost the same from infected and uninfected materials. A number of nucleotide polymerizing and nucleolytic activities were revealed. A DNA-directed RNA polymerase was demonstrated as well as three homopolymerase activities. Incorporations of ATP and CTP, requiring a primer (preferentially tRNA), were found in the same fractions and were probably caused by the same enzyme. An incorporation of GTP using poly(G) as primer likewise was detected. These polymerizations were inhibited in the presence of 10 mm pyrophosphate while 100 mm orthophosphate was not inhibitory. Furthermore, two peaks of nuclease activities have been found. The slow-eluting peak indicates an enzyme (enzymes) of approximately the same size as pancreatic ribonuclease (M_r = 13,680). The fast-eluting peak corresponds to molecular weights of the order of 150,000.     

RNA-synthesizing enzymes in healthy and TMV-infected tobacco leaves. Partial purification and characterization to tobacco polynucleotide phosphorylase

Polynucleotide phosphorylase was partially purified from healthy and TMV-infected tobacco leaves. The enzyme catalyzed all three typical polynucleotide phosphorylase activities. It required Mg²⁺ or Mn²⁺ for the polymerization reaction and was stimulated by NH⁺₄, K⁺, sulfhydryl agents, and polyamines. The synthetic activity was enhanced in the presence of a primer and was completely inhibited by 0.1 mm inorganic orthophosphate. The enzyme was insensitive to freezing. An approximate molecular weight of 150,000 was estimated.     

AXanthomonas maltophilia isolate tolerating up to 1% sodium azide in Tris/HCl buffer

A bacterium tolerating up to 1% NaN₃ found as a contaminant of Sephadex colums being run with Tris/HCl buffer, was identified asXanthomonas maltophilia. It had low nutrient requirements, was strongly proteolytic and interfered with Sephadex columns run with Tris/HCl buffers.The authors are with the Department of Plant Pathology, Swedish University of Agricultural Sciences, Box 7044, S-750 07 Uppsala, Sweden;     

Amino acid analysis of phloem sap from oats and barley: A combination of aphid stylet excision and high performance liquid chromatography

High performance liquid chromatography was used for amino acid analysis of phloem sap from oats and barley. The sap was obtained from severed stylets of the aphid Rhopalosiphum padi (L.) excised by means of high frequency microcautery. Qualitative differences between the cereals were negligible. The proportions of different amino acids largely resembled those demonstrated for whole leaf extracts. Samples gave very weak detector responses when dissolved in water and kept at room temperature. Explanations for this are discussed and a technique to prevent this deterioration is described.

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Résumé Au cours d'un programme de sélection contre Rhopalosiphum padi L. nous avons utilisé la micro-cautérisation à haute fréquence et la chromatographie liquide à haute performance pour obtenir de la sève du phloème de variétés sensibles d'avoine et d'orge, et pour analyser les acides aminés libres d'échantillons réduits (nl) de sève. Les résultats ont montré des teneurs en acides aminés libres plus élevées dans l'avoine que dans l'orge. Il n'y avait pas de différences qualitatives entre les deux céréales et les proportions en différents acides aminés correspondaient à celles obtenues par extraits totaux de feuilles. Quand les échantillons ont été dissous dans l'eau, les réponses obtenues étaient très faibles et une nouvelle technique a été utilisée pour y palier. L'utilité de cette méthode dans l'étude de l'écologie alimentaire des insectes suçeurs de sève est indiquée.