The transport of sugars between pea and powdery mildew

Aked, Julia (1990) The transport of sugars between pea and powdery mildew. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

A method has been developed for extracting washing fluid from the intercellular spaces of healthy and infected pea leaves. Measurements of sugar in this fluid showed that glucose and possibly fructose concentrations increased in the apoplast of infected leaves whereas sucrose levels were highly variable. Acid invertase activity in the washing fluid was very low and undetectable in pea cell wall fractions and whole leaf disks floated on sucrose solutions. Attempts to investigate symplastic and apoplastic pathways in infected pea leaves using lucifer yellow CH are described. The characteristics of sugar uptake into the lower epidermis of the pea mutant cv. argenteum were investigated using a leaf disk system. Uptake kinetics for glucose, fructose and sucrose were determined. Glucose has the highest V_max at 67.5 nmol dm^-2 h^-1 and sucrose has the lowest K_m at 0.04 mol m^-3. The effects of pH and various reagents such as parachloromercuribenzene sulphonic acid, erythrosin B and carbonyl cyanide m-chlorophenylhydrazone on sugar uptake were studied. Glucose uptake showed an optimum at pH 4.5. The specificity of putative sugar carriers on the epidermal plasma membrane was studied with a series of competition experiments. Glucose strongly competed with fructose and sucrose whereas fructose was a weaker competitor against glucose and sucrose. Sucrose was not a competitor for hexose uptake. Protoplasts were isolated from pea leaf tissue and epidermis removed from cv. argenteum leaves. An uptake system for sugars was established. The uptake of glucose and sucrose into mesophyll protoplasts appeared to result from leakage across damaged membranes and was insensitive to metabolic inhibitors. Epidermal sugar uptake was specific with sucrose entering at a slower rate than glucose. A leaf disk system was also used to study transport characteristics of sugars into the mycelium of Erysiphe pisi. Sucrose was taken up markedly slower than the hexoses at external concentrations above 5 mol m^-3. The uptake from 1 mol m^-3 glucose accelerates over a three hour period and is far higher than that from 1 mol m^-3 3-O-methylglucose. This suggests that glucose is rapidly metabolised by the mildew and that some aspect of glucose metabolism is switched on in response to the glucose. The effects of various metabolic inhibitors, pH and temperature on mycelial sugar uptake were studied. The results of these experiments were compared with those from epidermal sugar transport studies and the nature of possible sugar transport proteins on the host-fungal interface i.e. the haustorial membranes, is discussed. An improved method for isolating haustorial complexes (hcs) from mildew infected cv. argenteum leaves was developed. A maximum hc viability of 53% for such preparations was achieved. Attempts to set up an system for uptake of sugars and the potentiometric dye 3,3'-dibutyloxacarbocyanine iodide into hcs are described.

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