Barry J. Thomas

Of genes and genomes, needles and haystacks: Blumeria graminis and functionality

SUMMARY Here, we consider the barley powdery mildew fungus, Blumeria graminis (DC Speer) f.sp. hordei (Marchal), and review recent research which has added to our understanding of the biology and molecular biology which underpins the asexual life cycle of this potentially devastating pathogen. We focus on the early stages of the host-pathogen interaction and report current understanding in the areas of leaf perception, fungal signal transduction and host-imposed oxidative stress management. Through this, it is becoming increasingly clear how closely and subtly both sides of the relationship are regulated. Collectively, however, this review highlights the high degree of complexity in working with an obligate parasite. Our experiences suggest that we would make more efficient progress towards understanding the basis of susceptibility and resistance to this true obligate biotroph if its genome sequence was available.

Leaf senescence in a non-yellowing mutant of Festuca pratensis: Proteins of photosystem II

The senescence of leaves is characterized by yellowing as chlorophyll pigments are degraded. Proteins of the chloroplasts also decline during this phase of development. There exists a non-yellowing mutant genotype of Festuca pratensis Huds. which does not suffer a loss of chlorophyll during senescence. The fate of chloroplast membrane proteins was studied in mutant and wild-type plants by immune blotting and immuno-electron microscopy. Intrinsic proteins of photosystem II, exemplified by the light-harvesting chlorophyll a/b-binding protein (LHCP-2) and D1, were shown to be unusually stable in the mutant during senescence, whereas the extrinsic 33-kilodalton protein of the oxygen-evolving complex was equally lable in both genotypes. An ultrastructural study revealed that while the intrinsic proteins remained in the internal membranes of the chloroplasts, they ceased to display the heterogenous lateral distribution within the lamellae which was characteristic of nonsenescent chloroplasts. These observations are discussed in the light of possible mechanisms of protein turnover in chloroplasts.

Release and visualization of the extracellular matrix of conidia of Blumeria graminis

The time course of release of an extracellular matrix (ECM) from conidia of the barley powdery mildew pathogen Blumeria graminis was investigated by scanning electron and light microscopy. Conidia released the ECM material preferentially onto a hydrophobic surface and release was detected within 20 s of contact of a conidium with the substratum. No such rapid release of an ECM could be detected when conidia were deposited onto the hydrophilic surface of clean glass. A limited amount of ECM was, however, released from conidia onto the hydrophilic surface of a hydrated cellulose membrane. A time study on hydrophobic plastic revealed that although the ECM was clearly present within the first hour after contact of the conidium with the substratum, it was much reduced in quantity after 12 h incubation. The ECM was demonstrated by micromanipulation and light microscopy to be present as a liquid at the contact interface of the conidium and the substratum. The ECM could not be detected by SEM beneath conidia on leaves, but light microscopy and micromanipulation demonstrated that the ECM could be detected beneath some conidia on the waxy surface of an epidermal strip taken from the abaxial surface of a barley leaf. The ECM material stained positively for protein and could not be removed from the conidium interface by vacuum desiccation in the scanning electron microscope indicating that the ECM is not simply water.

Starch metabolism in the fructan-grasses: Patterns of starch accumulation in excised leaves of Lolium temulentum L.

Summary Excised and illuminated leaves of temperate gramineae have previously provided a powerful system for the analysis of the regulation of partitioning of photosynthate between sucrose and fructan. This paper complements previous data by describing the concomitant accumulation of transitory starch, an assimilate which has been ignored in this system. Leaves of Lolium temulentum were depleted to near-zero reserve carbohydrate content. In this condition, the tissue contained no detectable fructan or starch and only a trace of sucrose. When subsequently excised and illuminated continuously, the accumulation of sucrose and starch commenced immediately and occurred simultaneously. Starch anabolism was constitutive and the polymer was apparently synthesised de novo . The maximal rate of starch accumulation, at 0.6 mg g −1 fresh mass, was 5-10-fold lower than that of sucrose. Fructan was also synthesised de novo and was induced after a lag of 8 h, after which rates of net sucrose and starch accumulation slowed, ceasing completely by 14-16 h. Whilst total non-structural carbohydrate concentration increased continuously, reaching 56 mg g −1 at 30 h, starch concentration was asymptotic and was limited to a maximum of only 7 mg g −1 reached at 14-16 h. Hence, net starch accumulation was not restricted by the continued production of photosynthate. The illumination period spanned 1.25 diurnal cycles. Despite continued photoassimilation, there was no resumption of net starch synthesis at the beginning of the second cycle and hence the limitation of net starch accumulation was not imposed by an endogenous circadian rhythmicity. When assimilate partitioning into fructan was abolished by transpirational feeding of cycloheximide, sucrose concentration increased 2.7-fold to 49 mg g −1 . This abnormally high concentration did not feedback to enhance starch accumulation. Transpirational feeding of 10 mmol/L mannose inhibited sucrose accumulation by c .90 %, but this inhibition was not accompanied by an increase net starch accumulation, rather, starch accumulation was inhibited by 60 %. Mannose caused the formation of maltose in the tissue. Starch synthesis, in common with sucrose and fructan syntheses, was shown to be localised predominantly in mesophyll cells where presumably the control of partitioning between these three assimilates is exercised. The low rates, restricted tissue concentration, sucrose-insensitivity and mannose-inhibition of starch synthesis differ from many species where starch is the primary reserve. Although the available information is limited, some of these features are in common with other fructan grasses. The low capacity for starch accumulation provides a possible explanation for the evolution of fructan synthesis in these species.

Proteolysis and cell death in clover leaves is induced by grazing

Summary. Programmed plant cell death is a widespread phenomenon resulting in the formation of xylem vessels, dissected leaf forms, and aerenchyma. We demonstrate here that some characteristics of programmed cell death can also be observed during the cellular response to biotic and abiotic stress when plant tissue is ingested by grazing ruminants. Furthermore, the onset and progression of plant cell death processes may influence the proteolytic rate in the rumen. This is important because rapid proteolysis of plant proteins in ruminants is a major cause of the inefficient conversion of plant to animal protein resulting in the release of environmental N pollutants. Although rumen proteolysis is widely believed to be mediated by proteases from rumen microorganisms, proteolysis and cell death occurred concurrently in clover leaves incubated in vitro under rumenlike conditions (maintained anaerobically at 39 °C) but in the absence of a rumen microbial population. Under rumenlike conditions, both red and white clover cells showed progressive loss of DNA, but this was only associated with fragmentation in white clover. Cell death was indicated by increased ionic leakage and the appearance of terminal deoxynucleotidyl transferase-mediated dUTP-nick-end-labelled nuclei. Foliar protein decreased to 50% of the initial values after 3 h incubation in white clover and after 4 h in red clover, while no decrease was observed in ambient (25 °C, aerobic) incubations. In white clover, decreased foliar protein coincided with an increased number of protease isoforms.

Changes in activities of acid invertase isoforms affect sugar accumulation and composition during ripening of tomato fruit

Summary The observation that the wild, red fruited tomato Lycopersicon pimpinellifolium has higher soluble solids than the cultivated variety L. esculentum may be related to differential controls on fruit carbohydrate metabolism. We therefore investigated the role of acid invertase (AI) in determining fruit sugar composition in these two species. At all stages of fruit development, the majority of AI was localized in the vacuole with about 10% of the total activity in the extra-cellular space. Four forms of soluble AI activity were identified. Isoform 1 persisted throughout development. Isoform 2 showed a strong relationship to total fruit sugar content in both species, suggesting that this isoform is the tiv1 gene product located in the vacuole, and that it plays a major role in determining fruit soluble solids. Isoform 3 was very high in L. pimpinellfolium but not in L. esculentum. We suggest that AI has a major role in determining fruit sugar accumulation in L. pimpinellifolium, but not in L. esculentum. Pulse-chase in vitro unloading experiments with [3h]-glucose showed that a substantial amount of [3h]-sucrose was present in the apoplast, suggesting an exchange of sugars between extra- and intra-cellular spaces. Hence ““futile cycles’’ of sucrose synthesis and degradation involving apoplastic AI can occur during fruit development.