Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H₂O₂-and o-dianisidine/H₂O₂-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.This work was supported in part by the scientific Research Foundaation (OTKA F 5082), the foundation for Hungarian science, a british council scolership to A.L.A and the U.K. Agricultural and food Reaserch council.     

Anatomical characteristics and antioxidant ability of Centaurea sadleriana reveals an adaptation towards drought tolerance

The lamina, main vein and peduncle anatomical properties of Centaurea sadleriana Janka plants from two populations, were examined using light and scanning electron microscopy. The indumentum was comprised of glandular and non-glandular trichomes of two types. The leaves were amphistomatic, isolateral, with strongly developed palisade tissue. Secretory ducts were observed along the phloem or sclerenchyma of large vascular bundles. Collenchyma alternated with chlorenchyma in the main vein and peduncle. Large groups of strongly lignified sclerenchyma were present along the phloem of peduncle vascular bundles. These features, together with thickened walls of epidermal cells and cuticle, numerous trichomes and thick-walled parenchyma in the perimedullar zone, were perceived as a xeromorphic peduncle structural adaptation. Non-enzymatic antioxidant compounds of phenolic origin were detected in small amounts and their respective content was higher in leaves compared to inflorescences. Compounds of phenolic orgin showed positive correlation with total potenial of antioxidant activity indicated by the DPPH assay. Greater total quantity of polyphenols and tannins was detected in leaves of plants from Zobnatica locality, while leaves of plants from Rimski Sanac were characterized by higher content of total flavonoids and proantocyanidins. Phytochemical analysis showed that dominant secondary biomolecules in inflorescences were phenolic pigments including anthocyanins and leucoanthocyanins, and free quinones in leaves.     

Functional plant cell wall design revealed by the Raman imaging approach

Using the Raman imaging approach, the optimization of the plant cell wall design was investigated on the micron level within different tissue types at different positions of a Phormium tenax leaf. Pectin and lignin distribution were visualized and the cellulose microfibril angle (MFA) of the cell walls was determined. A detailed analysis of the Raman spectra extracted from the selected regions, allowed a semi-quantitative comparison of the chemical composition of the investigated tissue types on the micron level. The cell corners of the parenchyma revealed almost pure pectin and the cell wall an amount of 38–49% thereof. Slight lignification was observed in the parenchyma and collenchyma in the top of the leaf and a high variability (7–44%) in the sclerenchyma. In the cell corners and in the cell wall of the sclerenchymatic fibres surrounding the vascular tissue, the highest lignification was observed, which can act as a barrier and protection of the vascular tissue. In the sclerenchyma high variable MFA (4°–40°) was detected, which was related with lignin variability. In the primary cell walls a constant high MFA (57°–58°) was found together with pectin. The different plant cell wall designs on the tissue and microlevel involve changes in chemical composition as well as cellulose microfibril alignment and are discussed and related according to the development and function.     

Production of oxalic acid by Penicillium oxalicum in culture and in infected yam tissue and interaction with macerating enzyme

Penicillium oxalicum produced the greatest amount of oxalic acid in liquid culture and in infected yam tissue within 2 days of inoculation, whereas production of cell wall-degrading enzymes reached a peak eight days after inoculation. Coupled with the production of oxalic acid was the lowering of the culture pH and of infected yam tissue. Oxalic acid was not produced simultaneously with endo-polygalacturonase (the macerating enzyme), but it acted synergistically with the enzyme by sequestering calcium present in the middle lamella of yam cells and conditioning the cell wall for hydrolysis.     

Glutelin accumulation and changes in the levels of its mRNA in the superior and inferior spikelets of rice ear during ripening

Glutelin accumulation in the apical spikelet of the top primary branch (superior spikelet) and the second spikelet of the lowest secondary branch (inferior spikelet) of the ear of the rice plant (Oryza sativa L.) was characterized during grain filling.In the superior spikelet, the accumulation of dry matter and nitrogen started immediately after flowering and rapidly reached the maturation level by 20 days after heading (DAH). At 7 DAH, total RNA content had already reached its maximum level and glutelin mRNA content 70% of its maximum. The increase in glutelin mRNA was followed by a rapid increase in glutelin between 7 and 16 DAH.In the inferior spikelet dry matter, nitrogen and glutelin accumulation were low immediately after flowering and increased only after grain filling of the superior spikelet was almost complete. Total RNA and glutelin mRNA increased much later at slower rates than in the superior spikelet.It is very likely that the retardation of dry matter, total nitrogen and glutelin accumulation in the inferior spikelet is due to retardation of differentiation and development of endosperm tissue, and to glutelin gene expression in endosperm cells. It is suggested that the delayed development resulted from limited partitioning of nutrients to the inferior spikelet at the early stage of ripening.     

The initiation of cell wall synthesis in parasynchronous cultures of Schizosaccharomyces pombe

Summary Schizosaccharomyces pombe has been grown in parasynchronous culture to study the synthesis of cell wall material. After a lag period of 2.5h following inoculation the cells began to grow, as measured by optical density, dry weight and cell size. The cell number remained constant until 4.5h after inoculation when approximately 70% of the population divided synchronously. Immunofluorescence studies of the growing cells have shown that new wall material is inserted at the cell apices from 2.5 h after inoculation; this result is supported by radio-isotope labelling data which indicated that synthesis of new cell wall material also commenced 2.5 h after inoculation. The incorporation experiments also demonstrated an interruption in cell wall synthesis during the cell separation stage. The composition of the cell wall material varied during the growth cycle, with maximum nitrogen levels at inoculation and following cell division. No serological differences could be detected in the cell walls during the growth cycle.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Hypersensitive response, cell death and histochemical localisation of hydrogen peroxide in host and non-host seedlings infected with the downy mildew pathogen Sclerospora graminicola

Hypersensitive response, cell death and release of hydrogen peroxide as measures of host and non‐host defense mechanisms upon inoculation with the downy mildew pathogen Sclerospora graminicola were studied histochemically at the light microscopy level. The materials consisted of coleoptile tissues of the highly susceptible (cv. HB3), highly resistant (cv. IP18293) and induced resistant pearl millet host seedlings and non‐host sorghum (cv. SGMN10/8) and cotyledon of french bean (cv. S9). Resistance up to 80% protection against the downy mildew pathogen was induced in the highly susceptible HB3 cultivar of pearl millet by treating the seeds with 2% aqueous leaf extract of Datura metel for 3 h. Time course study with the pathogen inoculated highly resistant pearl millet cultivar revealed the appearance of hypersensitive response in 20% of seedlings as necrotic spots as early as 2 h after inoculation. In contrast, a similar reaction was observed in the highly susceptible pearl millet cultivar only 8 h after inoculation with the pathogen. In induced resistant seedlings, appearance of hypersensitive response was recorded 4 h after inoculation. Delayed hypersensitive response was observed in both the non‐host species at 10 h after inoculation. Hypersensitive response in the seedlings of the highly resistant pearl millet cultivar 24 h after inoculation showed 100% hypersensitive response, which was not observed in susceptible and non‐host species, although the induced resistant seedlings showed 90% hypersensitive response after that period of time. Cell death in the tissues of the test seedlings was also observed to change with time. Statistical analysis revealed that the tissues of highly resistant pearl millet seedlings required 2.9 h to attain 50% cell death. Tissues of induced resistant and highly susceptible pearl millet seedlings required 4.65 and 6.50 h respectively. In non‐hosts, 50% cell death was not recorded. Quantification of hydrogen peroxide in the tissue periplasmic spaces of the test seedlings revealed 2.94 h as the time required for 50% hydrogen peroxide accumulation in the tissues of highly resistant pearl millet seedlings. Tissues of induced resistant and highly susceptible pearl millet seedlings needed 3.76 and 5.5 h respectively. Fifty percent hydrogen peroxide localisation in non‐hosts could not be recorded. These results suggested the involvement of hydrogen peroxide, cell death and hypersensitive response in pearl millet host defense against S. graminicola.     

EVOLUTION OF LEAF ARCHITECTURE IN THE CHAPARRAL SPECIES FREMONTODENDRON CALIFORNICUM SSP. CALIFORNICUM (STERCULIACEAE)

The chaparral species Fremontodendron californicum ssp. californicum and the cloud forest species Cheirostemon platanoides arose from a common mesophytic stock. The leaf of C. platanoides is proposed as a model for the ancestral leaf and provides an architectural baseline allowing identification of the types and relative magnitudes of morphological and anatomical modifications which have occurred in the leaf of F. californicum. The leaf of F. californicum reflects intense selection for reduced surface area; however, both deletion and addition of lobes has occurred. Vein density has increased due to a greater number of more highly branched terminal veinlets. Mechanical structures, i.e., sclerenchyma, cuticle, show a quantitative reduction; whereas, water storage tissue, i.e., mucilage cells, has been augmented at the expense of spongy mesophyll. The leaf has become weakly isolateral and the number of palisade strata has increased. No increase in the volume of palisade tissue per unit volume of mesophyll is apparent due to a reduction in palisade cell length and palisade packing density. Total leaf thickness has not increased. The indumentum shows selection for non-living, pedestalled stellate trichomes which form a transpiration-limiting canopy above the stomata. Stomatal density has decreased while guard cell dimensions have become larger. Some structural modifications in F. californicum appear to have evolved directly in response to long-term water stress, others seem more directly related to leaf diminution. Xeroplastic changes in foliar structure are in some cases unreliable indicators of ultimate xeromorphic modifications arising in an evolutionary context.     

Production of Pectic Enzymes by Fusarium oxysporum f. sp. cepae and its Involvement in Onion Bulb Rot

Fusarium oxysporum f. sp. cepae produced an exo-polygalacturonase (exo-PG) and endopectin-trans-eliminase (endo-PTE) in a mineral medium supplemented with a restricted supply of either D-galacturonic acid or onion cell walls. These enzymes were also extracted from infected onion tissue, but only endo-PTE caused tissue maceration and cell death. The patterns of host tissue colonization and pectic enzyme production were followed during bulb rot development. Stem plates were invaded within two weeks of inoculation. The pathogen then remained confined to the stem plates for several weeks or months, before spreading to the outer fleshy scales to initiate a basal rot. In most cases the inner leaf sheaths containing the lateral bud remained healthy. Exo-PG activity m stem plate tissue was greatest at two weeks after inoculation, then it declined. Endo-PTE was not detected in newly invaded stem plate tissue, but was recovered from infected stem plates before decay and from the bases of bulb scales and leaf sheaths at the onset of bulb rot. There was no pectic enzyme activity in uninvaded onion tissue. Spread of the fungus and pectic enzyme production in two Caledon Globe genotypes susceptible or tolerant to F. oxysporum f. sp. cepae were similar, but the onset of bulb rot in tolerant genotypes was considerably delayed.     

Ultrastructural and cytochemical studies on the infection of wheat spikes by<Emphasis Type="Italic">Fusarium culmorum</Emphasis> as well as on degradation of cell wall components and localization of mycotoxins in the host tissue

The infection process and pathway of spreading ofFusarium culmorum in wheat spikes was examined by means of light, scanning and transmission electron microscopy after spray inoculation and single spikelet inoculation. Macroconidia of the pathogen germinated on the host surfaces, however, hyphal development and penetration of host tissues normally occurred on the inner surfaces of the lemma, glume and palea as well as on the ovary. The pathogen spread downward to the rachilla and rachis node by inter- and intracellular growth from the glume, lemma, palea and ovary. The pathogen extended in the rachis in upward and downward direction by inter- and intracellular growth inside and outside of the vascular bundles of the rachis. The spreading of the hyphae in the host tissues was associated with pronounced alterations including disintegration and digestion of host cell walls, suggesting production of cell wall degrading enzymes during infection and spreading in the host tissues. Immunogold labelling studies revealed that accumulation ofFusarium toxins in infected wheat spike tissue showed a close relationship to pathological changes in the host cells, symptom appearance and pathogen colonisation of the host tissue.Fusarium toxins may play an important role in wheat head blight development.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

A time-course study of early establishment stages of parasitic angiosperm Striga asiatica on susceptible sorghum roots

A time-course study of the early establishment stages of Striga asiatica was carried out on a susceptible sorghum hybrid, CSH 1, using polyethylene bags and whole-root clearing and staining techniques. Preconditioned Striga seeds were applied to different aged segments of primary root but the results did not differ for these different aged segments. Most of the Striga seeds (63%) germinated within 24 h of inoculation on the host roots. The attachment of Striga radicles to host root was rapid and it occurred between 36 and 48 h after inoculation. Only 9% of the germinated Striga seeds attached to the host root but 65% of these attachments successfully penetrated through the epidermis and entered the host cortex within 72 h. Penetration through the cortical cells was difficult; only 17% of attachments were able to reach the endodermis. Penetration took from 12 to 43 h after the first appearance of haustorial cells in the cortex; a total of 84 to 120 h after inoculation on the host root. Penetration through the endodermis and establishment on the host stele was relatively easier, as most of the haustoria reaching the endodermis were able to establish on the host stele. But this is a slow process taking a minimum of 24 h, and a maximum of 60 h after first contact of haustorial cells with the endodermis. The minimum time taken from inoculation of ungerminated Striga seed on the host root to establishment is about 108 h. The results are discussed in relation to published reports on other parasitic species such as Agalinis purpurea.     

贺兰山10种不同植物的旱生结构研究

对贺兰山10种不同生活型植物的形态解剖研究表明,长期生长在干旱贫瘠环境中植物,均形成适应生境的一些特殊形态结构．叶器官主要表现在：（1）叶表面积与体积之比缩小,表面覆有厚的角质膜,并被有表皮毛,气孔下陷,具孔下室;（2）栅栏薄壁组织细胞明显增多,海绵薄壁组织细胞减少．有叶肉不分化叶、等而叶和异而叶3种结构形式;（3）叶内贮水组织和机械组织增强。轴器官主要表现在：（1）根普遍形成周皮,且木栓层细胞层数增多;（2）机械组织非常发达,在周皮、皮层和韧皮部中有许多厚壁细胞分布。维管组织中的木薄壁细胞的细胞壁也明显木质化加厚;（3）有些植物的根具有异常维管组织。另外,这些植物根、茎、叶中均有粘液细胞和含晶细胞分布。这些结构具有重要的适应干旱生境的意义。     

Ultrastructure of the Water-absorbing Trichomes of Pineapple (Ananas comosus, Bromeliaceae)

An ultrastructural study of dry and wet trichomes from the upperleaf surface of pineapple revealed that shield cells were normallydead and stalk cells were often living. In stalk cells of drytrichomes, polysaccharide material was present within vacuolesand a layered material was present between the plasma membraneand cell wall. In stalk cells of trichomes wet for 12 h vacuoleswere enlarged and appeared to contain little material. Layeredmaterial between the plasma membrane and cell wall was alsoabsent. In both wet and dry trichomes, numerous mitochondriawere present and plasmodesmata were observed connecting stalk,foot, and adjacent epidermal and mesophyll cells. It is suggestedthat the large numbers of mitochondria, which indicate highmetabolism, and the numerous plasmodesmata connecting the cells,provide indirect evidence for the function of the trichomesin uptake of dissolved nutrients. Ananas comosus (L.) Merr., pineapple, trichomes, ultrastructure, Bromeliaceae     

Detection of the 3a Protein of Cucumber Mosaic Virus in a Cell Wall Fraction from Infected Nicotiana clevelandii Plants

The 3a protein of cucumber mosaic virus was expressed in Escherichia coli and, after purification, used to produce an antiserum. The 3a protein was detected in a cell wall fraction obtained from infected Nicotiana clevelandii leaf tissue by immunoblotting using the 3a antiserum. The 3a protein reached a maximum level 4 days after inoculation and remained at this level for a further 8 days before slowly declining. In contrast, the virus capsid protein, detected in an 80 000 g pellet by immunoblotting using a virus particle antiserum, reached a maximum 5 days after inoculation and remained at this level for at least a further 16 days.     

Histological and Ultrastructural Studies on the Curative Effects of Mandipropamid on Plasmopara viticola

The mandelic acid amide, mandipropamid, which belongs to the carboxylic acid amide (CAA) fungicides, is active against Plasmopara viticola, the causal agent of grapevine downy mildew. The fungicide primarily inhibits the germination of encysted zoospores, thus preventing the pathogen’s penetration into the host tissues, but it also shows curative effects. In this study, the infection structures of P. viticola in both leaves and berries were investigated to detect the histological and ultrastructural alterations induced by mandipropamid when applied after inoculation. Compared to the untreated samples characterized by a diffuse colonization of the tissues and by a normal ultrastructure of the pathogen, the application of mandipropamid 24 h after inoculation with P. viticola reduced pathogen colonization in leaves and berries. In addition, detachment of the plasmalemma from the hyphal and haustorial walls was observed 72 h after inoculation. In the berries, an abnormal proliferation of the pathogen plasma membrane was observed. Collapsed hyphae and haustoria in treated leaves were surrounded by callose or encapsulated in an amorphous material inside the host cell 72 h after inoculation, while a similar effect was observed in later stages (7 days) in berries. The results confirm that mandipropamid, which acts at the interface between the pathogen plasmalemma and cell wall, has curative activity against P. viticola, appearing more rapidly in leaves than in berries.     

线纹香茶菜叶上腺毛发育的细胞学研究

为进行中药溪黄草基原植物的品种鉴定,采用光镜和电镜对线纹香茶菜(原变种)[Isodon lophanthoides var.lophanthoides]叶上腺毛的发育进行细胞学研究。结果表明,线纹香茶菜具有头状腺毛和盾状腺毛2种类型。头状腺毛无色透明,由1个基细胞、1个柄细胞和1或2个头部分泌细胞构成;盾状腺毛为红色,由1或2个基细胞、1个柄细胞和4~8个分泌细胞构成头部。2种腺毛均由原表皮细胞经两次平周分裂形成,后因柄细胞和头部细胞所处的分化状态不同而形成两类腺毛。2种腺毛超微结构表明,质体、高尔基体和粗面内质网为主要分泌物产生和运输的细胞器。当盾状腺毛成熟时,角质层下间隙充满了分泌物,其分泌物的性质很可能决定了线纹香茶菜腺毛的颜色。