Active oxygen species in pea seedlings during the interactions with symbiotic and pathogenic microorganisms

The level of active oxygen species (AOS)--superoxide anion radical (O2*-) and hydrogen peroxide (H2O2)--in pea (Pisum sativum L.) cultivar Marat seedlings was studied upon their inoculation with symbiotic (Rhizobium leguminosarum bv. viceae strain CIAM 1026) and pathogenic (Pseudomonas syringae pv. pisi Sackett) microorganisms. Different patterns of the changes in AOS in pea seedlings during the interactions with the symbiont and the phytopathogen were recorded. It is assumed that O2*- and H2O2 are involved in the defense and regulatory mechanisms of the host plant.     

Application of Laser Microdissection to plant pathogenic and symbiotic interactions

Abstract

Laser Microdissection (LM) is a technology that allows the rapid procurement of selected cell populations from a section of heterogeneous tissues in a manner conducive to the extraction of DNA, RNA, proteins and even metabolites. In the past few years, it has also been applied to plant biology in order to study gene expression in plant-nematode and plant-microbe interactions. LM represents a powerful tool since cells associated with a particular infection stage can be visualized under the microscope and harvested. Therefore, verification of the response of the plant during the progression of the colonization can be performed in different cell types. Applications of LM to study the interaction between the plant and both pathogenic and symbiotic organisms (i.e. nematode and fungi, respectively) are explored in this review.     

Reactive oxygen species in programmed death of pea guard cells

Hydrogen peroxide potentiates CN(-)-induced apoptosis of guard cells recorded as destruction of cell nuclei in the epidermis from pea leaves. A still stronger effect was exerted by the addition of H2O2 and NADH, which are the substrates of the plant cell wall peroxidase producing O2*- coupled to the oxidation of NADH. The CN(-)-or (CN(-) + H2O2)-induced destruction of guard cell nuclei was completely removed by nitroblue tetrazolium (NBT) oxidizing O2*- and preventing there-by the subsequent generation of H2O2. The reduced NBT was deposited in the cells as formazan crystals. Cyanide-induced apoptosis was diminished by mannitol and ethanol, which are OH* traps. The dyes Rose Bengal (RB) and tetramethylrhodamine ethyl ester (TMRE) photosensitizing singlet oxygen production suppressed the CN(-)-induced destruction of the cell nuclei in the light. This suppression was removed by exogenous NADH, which reacts with 1O2 yielding O2*-. Incubation of leaf slices with RB in the light lowered the photosynthetic O2 evolution rate and induced the permeability of guard cells for propidium iodide, which cannot pass across intact membranes. Inhibition of photosynthetic O2 evolution by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea or bromoxynil prevented CN(-)-induced apoptosis of guard cells in the light but not in the dark. RB in combination with exogenous NADH caused H2O2 production that was sensitive to NBT and estimated from dichlorofluorescein (DCF) fluorescence. Data on NBT reduction and DCF and TMRE fluorescence obtained using a confocal microscope and data on the NADH-dependent H2O2 production are indicative of generation of reactive oxygen species in the chloroplasts, mitochondria, and nuclear region of guard cells as well as with participation of apoplastic peroxidase. Cyanide inhibited generation of reactive oxygen species in mitochondria and induced their generation in chloroplasts. The results show that H2O2, OH*, and O2*- resources utilized for H2O2 production are involved in apoptosis of guard cells. It is likely that singlet oxygen generated by RB in the light, judging from the permeability of the plasmatic membrane for propidium iodide, makes Photosystem II of chloroplasts inoperative and induces necrosis of the guard cells.     

Study of morphological and symbiotic traits during the ontogeny of supernodular and hypernodular pea mutants

Morphological (plant height and vegetative biomass amount) and symbiotic (number of nodules and nitrogenase activity) traits of six symbiotic pea mutants and the original cultivar Rondo were studied at different vegetation periods. Of the mutants studied, one (K10a) was supemodular and the remaining five (K1a, K2a, K5a, K7a, and K27a) were hypemodular. Essential distinctions in the absolute values and time course of the changes in individual morphological and symbiotic traits of different pea mutants were demonstrated. The supemodular type is inferior to the original cultivar in plant height and production of vegetative biomass, but exceeds it in nodulation and nitrogen fixation. The hypemodular mutants either surpass the original cultivar with respect to the production capacity or display similar results. The symbiotic traits-number of nodules and nitrogen fixation activity--of these mutants are higher compared with the Rondo cultivar. The mutants K1a, K2a, and K27a were demonstrated to be useful in breeding pea for an increase in nitrogen fixation.     

Study of morphological and symbiotic traits during the ontogeny of supernodular and hypernodular pea mutants

Morphological (plant height and vegetative biomass amount) and symbiotic (number of nodules and nitrogenase activity) traits of six symbiotic pea mutants and the original cultivar Rondo were studied at different vegetation periods. Of the mutants studied, one (K10a) was supernodular and the remaining five (K1a, K2a, K5a, K7a, and K27a) were hypemodular. Essential distinctions in the absolute values and time course of the changes in individual morphological and symbiotic traits of different pea mutants were demonstrated. The supernodular type is inferior to the original cultivar in plant height and production of vegetative biomass, but exceeds it in nodulation and nitrogen fixation. The hypernodular mutants either surpass the original cultivar with respect to the production capacity or display similar results. The symbiotic traits—number of nodules and nitrogen fixation activity—of these mutants are higher compared with the Rondo cultivar. The mutants K1a, K2a, and K27a were demonstrated to be useful in breeding pea for an increase in nitrogen fixation.     

Density-dependent interactions between seedlings of Dactylorhiza majalis (Orchidaceae) in symbiotic in vitro culture

Growth of the heterotrophic seedlings of Dactylorhiza majalis (Rchb.f.) Hunt & Summerh. (Orchidaceae) in vitro with a symbiotic fungus ( Rhizoctonia sp.) is density-dependent, even at densities that are not high enough to exploit the growth medium fully. Competition was two-sided; increasing density among seedlings did not increase size inequality between them. The slowly growing and smaller seedlings are normally more likely to die than the bigger ones, but mortality was not increased by higher density within the studied range. Growth depression by neighbouring seedlings is independent of their physical distance but varies with the total number of seedlings sharing the same culture dish and the same mycelium.
Therefore, if the growth-promoting effects of different fungi or treatments are to be compared in future work, the density of planting in the culture dishes should be comparable.     

Effect of electron-transport inhibitors on the generation of reactive oxygen species by pea mitochondria during succinate oxidation

The effect of inhibitors of the cytochrome pathway and alternative oxidase on the rate of respiration and generation of reactive oxygen species by pea mitochondria was studied. Respiration of mitochondria from pea cotyledons was inhibited by 70-80% by salicylhydroxamate (SHAM). The rate of hydrogen peroxide production by pea cotyledon mitochondria during succinate oxidation was 0.15 nmol/min per mg protein. SHAM considerably accelerated the hydrogen peroxide production. The SHAM-dependent H₂O₂ production was stimulated by 2 M antimycin A and inhibited by 5 mM KCN and 1 M myxothiazol. The study of the rate of generation by pea mitochondria using EPR spin traps and epinephrine oxidation showed that H₂O₂ accumulation can be accounted for by a significant increase in the rate of production.     

Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts*

Gram-negative bacteria communicate with each other by producing and sensing diffusible signaling molecules. This mechanism is called quorum sensing (QS) and regulates many bacterial activities from gene expression to symbiotic/pathogenic interactions with hosts. Therefore, the elucidation and control of bacterial QS systems have been attracted increasing attention over the past two decades. The most common QS signals in Gram-negative bacteria are N-acyl homoserine lactones (AHLs). There are also bacteria that employ different QS systems, for example, the plant pathogen Ralstonia solanacearum utilizes 3-hydroxy fatty acid methyl esters as its QS signals. The QS system found in the endosymbiotic bacterium associated with the fungus Mortierella alpina, the development of an affinity pull-down method for AHL synthases, and the elucidation of a unique QS circuit in R. solanacearum are discussed herein.     

Active oxygen species as factors in multistage carcinogenesis.

Oxygen, a necessary element for the life of a cell, is also the source of active states of oxygen including radicals, which can disrupt cell structure and alter cell function. Increasing evidence indicates that active oxygen species are formed in response to tumor promoters and that the cellular consequences of their actions may play a role in the process of tumor promotion. This report summarizes work from our laboratory that implicates active oxygen species derived in part from phagocytic cells in the tumor promotion process by phorbol esters and other promoters in mouse skin. Work from other laboratories indicates that phorbol ester promoters stimulate the production of active states of oxygen in mouse skin epidermal cells in vivo and in vitro. Oxidative DNA damage in epidermal cells from mice treated topically with the potent promoter phorbol myristate acetate has also been reported. The production of active states of oxygen including free radicals is discussed in relation to the mode of action of complete, first, and second stage promoters in the multistage carcinogenesis model in mouse skin.     

Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria

The effect in vivo of salt stress on the activated oxygen metabolism of mitochondria, was studied in leaves from two NaCl-treated cultivars of Pisum sativum L. with different sensitivity to NaCl. In mitochondria from NaCl-sensitive plants, salinity brought about a significant decrease of Mn-SOD (EC 1. 15. 1. 1) Cu, Zn-SOD I (EC 1. 15. 1. 1) and fumarase (EC 4. 2. 1. 2) activities. Conversely, in salt-tolerant plants NaCl treatment produced an increase in the mitochondrial Mn-SOD activity and, to a lesser extent, in fumarase activity. In mitochondria from both salt-treated cultivars, the internal H₂O₂ concentration remained unchanged. The NADH- and succinate-dependent generation of O₂^.−radicals by submitochondrial particles and the lipid peroxidation of mitochondrial membranes, increased as a result of salt treatment, and these changes were higher in NaCl-sensitive than in NaCl-tolerant plants. Accordingly, the enhanced rates of superoxide production by mitochondria from salt-sensitive plants were concomitant with a strong decrease in the mitochondrial Mn-SOD activity, whereas NaCl-tolerant plants appear to have a protection mechanism against salt-induced increased O₂^.− production by means of the induction of the mitochondrial Mn-SOD activity. These results indicate that in the subcellular toxicity of NaCl in pea plants, at the level of mitochondria, an oxidative stress mechanism mediated by superoxide radicals is involved, and also imply a function for mitochondrial Mn-SOD in the molecular mechanisms of plant tolerance to NaCl.     

昆虫共生微生物及其功能研究进展

昆虫体内共生微生物能够占到昆虫生物量的1％～10％,主要包括细菌、真菌、古菌和病毒.昆虫与共生微生物共进化形成共生体,共生微生物在昆虫生物学性状、多样性形成、生态适应性与抗逆性等多方面发挥着重要的作用.昆虫中的农作物害虫严重影响农业生产.本文对2000年以来农业害虫共生微生物的多样性、研究方法和功能机制、共生微生物之间...     

Relationship of symbiotic microorganisms to metanephridium: Phagoctic activity in the metanephridial epithelium of two species of oligochaeta

It is known that a number of species in the annelid family Lumbricidae harbor symbiotic microorganisms in the lumen of their nephridia. The purpose of the present paper is the study of the relationship between microbes and epithelial cells lining the metanephridium of two species of Oligochaeta, which show two different patterns of microbial colonization. A new interesting feature, the phagocytosis and intracellular destruction of microorganisms by the nephridial epithelial cells, has been observed in our laboratory for the first time. In Scheroteca savignyi minor, the phagocytic activity takes place in the bladder, the most distal region of the nephridium, next to the nephridiopore, which may prevent the microorganisms from entering the more proximal regions. In Octolasion cyaneum the microbes reach the striated duct, where they live in symbiosis, adhere to the cell's surface, and are engulfed and destroyed by the cells of the middle tubule—the more proximal, neighboring region. The phagocytosis and intracellular degradation of microorganisms probably lead to the massive formation of lamellar bodies, which are observed in these cells and in the neighboring ones.     

The phytotoxin coronatine induces light-dependent reactive oxygen species in tomato seedlings

The phytotoxin coronatine (COR), which is produced by Pseudomonas syringae pv. tomato DC3000 (DC3000), has multiple roles in virulence that lead to chlorosis and a reduction in chlorophyll content. However, the physiological significance of COR-induced chlorosis in disease development is still largely unknown. Global expression analysis demonstrated that DC3000 and COR, but not the COR-defective mutant DB29, caused reduced expression of photosynthesis-related genes and result in a 1.5- to 2-fold reduction in maximum quantum efficiency of photosystem II (F(V)/F(M)). Tomato (Solanum lycopersicum) seedlings inoculated with DC3000 and incubated in a long daily photoperiod showed more necrosis than inoculated seedlings incubated in either dark or a short daily photoperiod. The accumulation of reactive oxygen species (ROS) was detected in cotyledons inoculated with either purified COR or DC3000 but not in tissues inoculated with DB29. Interestingly, COR-induced ROS accumulated only in light and was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea and diphenylene iodonium, which function to inhibit electron transport from PSII. Furthermore, COR and DC3000 suppressed expression of the gene encoding the thylakoid Cu/Zn superoxide dismutase but not the cytosolic form of the same enzyme. In conclusion, these results demonstrate a role for COR-induced effects on photosynthetic machinery and ROS in modulating necrotic cell death during bacterial speck disease of tomato.     

Gibberellin-like activity in pea seedlings during growth under red,blue and white light

The influence of blue, red and white light and gibberellic acid (GA₃) on gibberellin-like activity in tissue extracts of leaves, stems and roots was investigated during growth of pea seedlings (Pisum salivum L. cv. Bördi). Higher GA-like activity was found in leaves and stems of pea plants that were growing in blue light than in those under red or white light. Patterns of change of activity were different in leaves, stems and roots, and in GA₃-treated plants.     

Metabolic and symbiotic interactions in amino acid pools of the pea aphid,Acyrthosiphon pisum,parasitized by the braconid Aphidius ervi

Aphidius ervi Haliday (Hymenoptera, Braconidae) is an endophagous parasitoid of the pea aphid, Acyrthosiphon pisum (Harris) (Homoptera, Aphididae). This parasitoid strongly redirects host reproduction and metabolism to favour nutrition and development of its juvenile stages. Parasite-regulated biosynthesis and mobilization of nitrogen metabolites determine a significant increase of host nutritional suitability. The aim of the present study was mainly to investigate the temporal changes of A. pisum amino acid pools, as affected by A. ervi parasitism, and to assess the role of the aphid bacterial endosymbiont Buchnera in determining the observed changes. In parasitized aphids, we observed a very significant increase in total free amino acids, compared with synchronous non-parasitized controls, starting from day 4 after parasitization (+51%). This trend culminated with more than doubling the control value (+152%) on day 6 after parasitization. However, a significant “parasitism” effect was observed only for 10 of the 28 amino acids detected. Tyrosine accumulation was the most prominent parasitoid-induced alteration, with a fourfold increase over control levels registered on day 6. In parasitized hosts, the amino acid biosynthetic capacity of Buchnera was unaltered, or even enhanced for the phenolic pool, and contributed greatly to the definition and maintainance of host free amino acid pools. The hypertyrosinemic syndrome was not dependent on food supply of the aromatic nucleus but was induced by parasitism, which likely enhanced the aromatic shuttle mediating phenylalanine transfer from bacteria to the host tissues, where tyrosine conversion occurs. This process is likely associated with a selective disruption of the host’s functions requiring tyrosine, leading to the remarkable accumulation of this amino acid. The possible mechanisms determining these parasitism-induced host alterations, and their nutritional significance for the developing parasitoid larva, are discussed.