Annerose Heller

Characterization of chromoplasts and carotenoids of red- and yellow-fleshed papaya (Carica papaya L.).

Chromoplast morphology and ultrastructure of red- and yellow-fleshed papaya (Carica papaya L.) were investigated by light and transmission electron microscopy. Carotenoid analyses by LC–MS revealed striking similarity of nutritionally relevant carotenoid profiles in both the red and yellow varieties. However, while yellow fruits contained only trace amounts of lycopene, the latter was found to be predominant in red papaya (51% of total carotenoids). Comparison of the pigment-loaded chromoplast ultrastructures disclosed tubular plastids to be abundant in yellow papaya, whereas larger crystalloid substructures characterized most frequent red papaya chromoplasts. Exclusively existent in red papaya, such crystalloid structures were associated with lycopene accumulation. Non-globular carotenoid deposition was derived from simple solubility calculations based on carotenoid and lipid contents of the differently colored fruit pulps. Since the physical state of carotenoid deposition may be decisive regarding their bioavailability, chromoplasts from lycopene-rich tomato fruit (Lycopersicon esculentum L.) were also assessed and compared to red papaya. Besides interesting analogies, various distinctions were ascertained resulting in the prediction of enhanced lycopene bioavailability from red papaya. In addition, the developmental pathway of red papaya chromoplasts was investigated during fruit ripening and carotenogenesis. In the early maturation stage of white-fleshed papaya, undifferentiated proplastids and globular plastids were predominant, corresponding to incipient carotenoid biosynthesis. Since intermediate plastids, e.g., amyloplasts or chloroplasts, were absent, chromoplasts are likely to emerge directly from proplastids.

Oxalic Acid Has an Additional, Detoxifying Function in Sclerotinia sclerotiorum Pathogenesis

The mechanism of the diseases caused by the necrotroph plant pathogen Sclerotinia sclerotiorum is not well understood. To investigate the role of oxalic acid during infection high resolution, light-, scanning-, transmission electron microscopy and various histochemical staining methods were used. Our inoculation method allowed us to follow degradation of host plant tissue around single hyphae and to observe the reaction of host cells in direct contact with single invading hyphae. After penetration the outer epidermal cell wall matrix appeared degraded around subcuticular hyphae (12-24 hpi). Calcium oxalate crystals were detected in advanced (36-48 hpi) and late (72 hpi) infection stages, but not in early stages. In early infection stages, surprisingly, no toxic effect of oxalic acid eventually secreted by S. sclerotiorum was observed. As oxalic acid is a common metabolite in plants, we propose that attacked host cells are able to metabolize oxalic acid in the early infection stage and translocate it to their vacuoles where it is stored as calcium oxalate. The effects, observed on healthy tissue upon external application of oxalic acid to non-infected, living tissue and cell wall degradation of dead host cells starting at the inner side of the walls support this idea. The results indicate that oxalic acid concentrations in the early stage of infection stay below the toxic level. In plant and fungi oxalic acid/calcium oxalate plays an important role in calcium regulation. Oxalic acid likely could quench calcium ions released during cell wall breakdown to protect growing hyphae from toxic calcium concentrations in the infection area. As calcium antimonate-precipitates were found in vesicles of young hyphae, we propose that calcium is translocated to the older parts of hyphae and detoxified by building non-toxic, stable oxalate crystals. We propose an infection model where oxalic acid plays a detoxifying role in late infection stages.

Ultrastructural deposition forms and bioaccessibility of carotenoids and carotenoid esters from goji berries (Lycium barbarum L.).

Goji berries (Lycium barbarum L.) have been known to contain strikingly high levels of zeaxanthin, while the physical deposition form and bioaccessibility of the latter was yet unknown. In the present study, we associated ripening-induced modifications in the profile of carotenoids with fundamental changes of the deposition state of carotenoids in goji berries. Unripe fruit contained common chloroplast-specific carotenoids being protein-bound within chloroplastidal thylakoids. The subsequent ripening-induced transformation of chloroplasts to tubular chromoplasts was accompanied by an accumulation of up to 36mg/100g FW zeaxanthin dipalmitate and further minor xanthophyll esters, prevailing in a presumably liquid-crystalline state within the nano-scaled chromoplast tubules. The in vitro digestion unraveled the enhanced liberation and bioaccessibility of zeaxanthin from these tubular aggregates in goji berries as compared to protein-complexed lutein from spinach. Goji berries therefore might represent a more potent source of macular pigments than green leafy vegetables like spinach.

Capitate glandular trichomes of Helianthus annuus (Asteraceae): ultrastructure and cytological development

Previous studies have shown that capitate glandular trichomes (CGT) of the common sunflower, Helianthus annuus, produce sesquiterpene lactones (STL) and flavonoids, which are sequestered and accumulated between the apical cuticle and the wall of the tip cells. To explore the cellular structures required and putatively involved in the STL biosynthesis and secretion, the present study was focused on the development of CGT and the comparison of the ultrastructure of its different cell types. Gradual maturation of flowers in the capitulum of the sunflower provided the possibility to study the simultaneous differentiation from the primordial to the secretory stage of CGT located by light microscopy (bright field, differential interference contrast and fluorescence) as well as transmission electron microscopy. It was shown that the CGT of sunflower anthers had a biseriate structure with up to 14 cell pairs. In mature trichomes, the apical cells called secretory cells were covered entirely by a large cuticle globe, which enclosed the resinous terpenoids and was specialised in thickness and structure. The secretory cells lacked chloroplasts and contained mainly smooth endoplasmic reticulum (sER). Conspicuous cell wall protuberances and an accumulation of mitochondria nearby occurred in the horizontally oriented cell walls. The cytological differences between stalk cells and secretory cells indicate a different function. The dominance of sER suggests its involvement in STL biosynthesis and cell wall protuberances enlarge the surface of the plasmamembrane of secretory cells and may be involved in the secretion processes of STL into the subcuticular space.

Evidence for the importance of enzymatic digestion of epidermal walls during subepidermal sporulation and pustule opening in white blister rusts (Albuginaceae)

Albugo candida, A. ipomoeae-panduratae, Pustula tragopogonis, Wilsoniana bliti and W. portulacae are widespread obligate biotrophic plant pathogens causing white blister diseases on a variety of flowering plants. Their subepidermal mode of sporulation is unique amongst Oomycetes and leads to blister-like structures on their hosts similar to those produced by true rusts (Uredinales). Unlike in true rusts, sporangia are colourless and produced in chains; the first formed, primary sporangium, differing in size and morphology from subsequent secondary sporangia. According to current interpretations of pustule development the rising pressure of the growing chains of sporangia tear off the epidermal layer from the mesophyll and, in the end, ruptures the epidermis to release the sporangia. This is not convincing considering the rigidity of the epidermal layer and the fact that thin-walled mesophyll cells show no signs of pressure endurance. Our detailed light-, scanning electron-, and transmission electron microscopic observations provide evidence that pustule development and opening are regulated and delicate processes that involve directed enzymatic dissection of host tissue cell walls. The process starts when intercellular hyphae separate the epidermal layer from the parenchyma, forming a cavity in which sporulation takes place. Then thick-walled sporogenous hyphae with club-shaped but thin-walled tips develop and produce sporangia in basipetal succession from the apices of the sporogenous hyphae. The short-living primary sporangia attach tightly to the inner cell walls of the epidermal layer and undergo dramatic cytological changes during pustule maturation, including vacuolisation and development of numerous electron-dense vesicles that might deliver cell wall degrading enzymes. In ripe pustules, the disintegration of areas of epidermal cells leads to the opening of the pustules and to the release of the secondary sporangia. Also the comparison of samples prepared for scanning electron microscopy with fresh pustules, as well as the comparison of the inner epidermal layers detached by the pathogens and detached by force supports our conclusion that delicate enzymatic activity and not force are involved in pustule development and opening by these highly sophisticated pathogens.

False-positive labeling of the haustorial cell wall of bean rust: a problem in immunogold labeling of thin sections

Abstract False-positive labeling was studied by immunogold-labeling methods. It was observed during an investigation of chitinase distribution in rust-infected bean leaves. Sixteen of the 23 antisera/immunoglobulins tested gave distinct and apparently specific labeling of the haustorial cell wall of the fungus, also with non-immune sera and non-related antisera: all of them were rabbit antisera or immunoglobulins. Only the three monoclonal and four of the rabbit sera (two non-immune and two antisera against plant viruses) showed weak or no reactions. The effect was demonstrated with antiserum against rubisco, which labeled the haustorical cell wall in rather high dilutions. There were no disturbing antibodies against fungi in the sera of the rabbits as suitable acetone powders were inefficient. The type of binding and which component of the cell wall is involved are unknown. It is speculated that there might be a similar binding effect as with protein A to certain mammal immunoglobulins. Blocking with normal goat serum reduced the effect but use of bovine serum albumin (BSA), gelatine and Tween 20 was ineffective. The localization experiment of chitinase is a case in point concerning the misinterpretation of the specificity of an immunogold-labeling test. Therefore, standard controls are by no means sufficient and the specificity has to be confirmed by multiple controls.

Seed Structure Characteristics of Orobanche cumana Populations

Abstract Sunflower broomrape Orobanche cumana Wallr. is a rapidly growing threat to the oil crop production in many countries. Fast adaptation to new environments and increasing host resistance suggests that phenotypically distinctive populations of the weed may have evolved. The classification of the species and the differentiation of such populations on the base of seed micromorphological characters were attempted. Morphometric measurements allowed the distinction of O. cumana from several other Orobanche and Phelipanche species. An irregularly thickened cell wall of the anticlinal testa cells differentiated O. cumana and O. cernua from O. caryophyllacea, O. crenata, O. minor, P. aegyptiaca, P. arenaria and P. ramosa. However, populations of sunflower broomrape from five European countries and China could not be separated from each other on the base of micromorphological seed characters. In contrast, length to width measurements indicated that the Asian samples had a slightly different seed shape which was less elongated than the European samples. However, this seemingly geographic effect may as well be a consequence of sampling which comprised a higher rate of the so-called modern races E-H in the European samples.

Localization of sesquiterpene lactone biosynthesis in cells of capitate glandular trichomes of Helianthus annuus (Asteraceae).

Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid.

Tissue specific reactions of sorghum roots to the mycoherbicide Fusarium oxysporum f. sp. strigae versus the pathogenic F. proliferatum

Abstract Fusarium oxysporum f. sp. strigae (Foxy 2) is a mycoherbicide against Striga hermonthica. To ensure the safe use of this biocontrol agent, and as part of the risk assessment, this study was aimed at providing cytological evidence that Foxy 2 does not possess pathogenic behavior towards the non-target host sorghum. Therefore, we compared the infection processes and sorghum root tissue reactions towards the pathogenic F. proliferatum to that of Foxy 2 using light- and transmission electron microscopy. Given that during the growth process, hyphae could get into the central cylinder, tissue specific reactions of sorghum to Foxy 2 were also investigated by wounding the roots (exposing the vascular system), and testing for proliferation of hyphae within the vessels. Results showed that 2 weeks after sowing, F. proliferatum had invaded and destroyed all cell types including the central cylinder while Foxy 2 hyphae were located around the outer endodermal layer and were not able to penetrate the latter. There was an increase in blue autoflourescence in the central cylinder and especially the endodermis, probably due to increased phenolics in Foxy 2 infected roots which was not the case for F. proliferatum. This might contribute to the inability of Foxy 2 to penetrate the endodermis. Transmission electron microscopy showed extensive degradation of endodermis and vessel walls into thin translucent layers by F. proliferatum but not by Foxy 2. In the mechanically wounded and infected roots, Foxy 2 could invade the central cylinder close to the wound but was not identified a few millimeters away from the wound. This implies that it was not able to grow within or destroy the central cylinder even when already present within it; probably due to the observed increased phenolics. Thus, exposure of the vascular system did not serve as a route for the invasion of Foxy 2 which therefore could not further cause tracheomycosis. Therefore, Foxy 2 could be seen as a fungus well suited for biocontrol.

Comprehensive Characterisation of n-Alkylresorcinols and Other Lipid Constituents of Mercurialis tomentosa L. from Alicante, Spain

Mercurialis tomentosa L. has been used in Spanish ethnomedicine. In the present study the first phytochemical characterisation of a lipid fraction from M. tomentosa was performed. The CHCl3 extraction of aerial parts from M. tomentosa and GC/MS investigations revealed the occurrence of cuticular lipid and wax constituents, like long chain n‐alcohols and n‐aldehydes (C22 – C30), besides several aromatic constituents, i.e., phenylpropanoids and n‐alkylresorcinols. The latter were further purified by CC and analysed by LC/MSn. In contrast to other Mercurialis species, i.e., M. annua, M. perennis, which exclusively contain 5‐n‐alkylresorcinols (1a – j, Cn), mainly 5‐n‐alkyl‐2‐methylresorcinols (2a – j, Cn*) with side chain lengths of C15 – C25 were found in M. tomentosa, in addition to 1a – j. Thus, the latter compounds may be utilised for analytical characterisation and authentication of M. tomentosa based on fingerprinting methods. For structure elucidation a novel facile total synthesis of one representative 5‐n‐alkyl‐2‐methylresorcinol homologue (2d, C19*) was developed, starting with a Grignard reaction from a substituted benzoic acid chloride (19). The compound obtained by synthesis was identical to the natural product 2d in terms of its chromatographic and spectroscopic features. Futhermore, 2d exhibited satisfactory DPPH free radical scavenging activity (IC50 = 37.8 μm) when compared to trolox (IC50 = 21.0 μm), corroborating the antioxidant features of these amphipathic molecules.