Merope Tsimilli-Michael

In vivo Assessment of Stress Impact on Plant’s Vitality: Applications in Detecting and Evaluating the Beneficial Role of Mycorrhization on Host Plants

Where land exists and the climate is supporting, life develops. Microorganisms, plants, animals and humans populate the site and they together form an ecosystem. Living together is a fact; the life quality of each individual is influenced by each member of the system, and no established life style will persist if the total ecosystem is not in a sustainable state. The maximization of exploitation of the resources by man leads in the long term, in all cases, to a disaster. Only optimization of the use of resources combined with recycling of waste and the regeneration of used areas can lead to sustainability. Every project based on a harmonic balance between the consumption and production of goods with respect towards the environment contributes to sustainability (Strasser and Tsimilli-Michael 1998). A tremendous amount of know-how is available and no method that can serve the purpose should be rejected. No problem of major importance can be solved by a single person. We have all to learn and to try how sustainability can be established. Every person is invited to participate in such projects with his knowledge and experience to build up a network of “Collective intelligence”. The work presented here aims to assess the vitality of plants, which is a prerequisite for sustainability and the conservation of biodiversity. Sustainable agriculture is today both a challenge and a necessity. More than a system of farming, it is a new approach integrating ecological, sociological, economical and even cultural aspects of life. The multidisciplinary character of this approach not only allows but requests the involvement of scientists from different fields, which will form a

Synergistic and antagonistic effects of arbuscular mycorrhizal fungi and Azospirillum and Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa, probed by the polyphasic chlorophyll a fluorescence transient O-J-I-P☆

The synergistic and antagonistic effects of arbuscular mycorrhizal fungi (AMF) and Azospirillum and Rhizobium nitrogen-fixers on the photosynthetic activity of alfalfa (Medicago sativa L.) were studied by means of the polyphasic chlorophyll a (Chl a) fluorescence transient O-J-I-P. The effects were evaluated on alfalfa plants grown in: (a) loamy chernozem control soil including the original rhizosphere community (bacteria+AMF); (b) gamma-sterilised soil (no microbes); (c) bacterial re-inoculated AMF-free soil (only bacteria). In these substrates the plants were inoculated with AMF (Glomus fasciculatum M107), with and without co-inoculation with the associative (Azospirillum brasilense Km5) and/or the symbiotic (Rhizobium meliloti Lu41+S5/7+K4/1) nitrogen-fixing bacteria. The Chl a fluorescence transients were recorded in vivo and analysed according to the JIP-test leading to the calculation of a constellation of parameters quantifying the photosystem II (PSII) behaviour. The beneficial effect of AMF is clearly revealed by the observed enhancement of the electron transport activity per leaf area. Based on the same criterion, an antagonism by both bacteria was detected. The antagonistic effect of Azospirillum was more pronounced than that of Rhizobium, though not strong enough to fully counter-balance for the beneficial effect of AMF. However, in the case of co-inoculation with both diazotrophs and AMF the electron transport activity was found to be only slightly lower than in the case of single inoculation by AMF, indicating that, in the presence of each other, the diazotrophs are no longer antagonistic to AMF. It was further clarified that these antagonistic effects are the net result of different synergistic and antagonistic effects, as follows: (a) Concerning the electron transport activity per absorption, Rhizobium has a synergistic effect on AMF’s beneficial role, which is even more pronounced in the case of the tripartite co-inoculation, though Azospirillum in the absence of Rhizobium is antagonistic to AMF. (b) Concerning absorption per leaf, the beneficial influence of AMF is almost fully counterbalanced by co-inoculation with each of the diazotrophs or by both of them. The results demonstrate that the different combinations of inoculations and/or soils can be well distinguished by means of the JIP-test parameters and they thus suggest that the test can be used to screen, through the PSII behaviour of the plants, the effect of the microbial activity in the field. Two-dimensional rankings of the several soil/microbe combinations, in respect to the values of different energy fluxes, are shown to provide mappings that can be useful for the comparison of whole ecosystems or of individuals within an ecosystem.

Response of endangered plant species to inoculation with arbuscular mycorrhizal fungi and soil bacteria

Three endangered plant species, Plantago atrata and Pulsatilla slavica, which are on the IUCN red list of plants, and Senecio umbrosus, which is extinct in the wild in Poland, were inoculated with soil microorganisms to evaluate their responsiveness to inoculation and to select the most effective microbial consortium for application in conservation projects. Individuals of these taxa were cultivated with (1) native arbuscular mycorrhizal fungi (AMF) isolated from natural habitats of the investigated species, (2) a mixture of AMF strains available in the laboratory, and (3) a combination of AMF lab strains with rhizobacteria. The plants were found to be dependent on AMF for their growth; the mycorrhizal dependency for P. atrata was 91%, S. umbrosus-95%, and P. slavica-65%. The applied inocula did not significantly differ in the stimulation of the growth of P. atrata and S. umbrosus, while in P. slavica, native AMF proved to be the less efficient. We therefore conclude that AMF application can improve the ex situ propagation of these three threatened taxa and may contribute to the success of S. umbrosus reintroduction. A multilevel analysis of chlorophyll a fluorescence transients by the JIP test permitted an in vivo evaluation of plant vitality in terms of biophysical parameters quantifying photosynthetic energy conservation, which was found to be in good agreement with the results concerning physiological parameters. Therefore, the JIP test can be used to evaluate the influence of AMF on endangered plants, with the additional advantage of being applicable in monitoring in a noninvasive way the acclimatization of reintroduced species in nature.

Optimization of culture conditions of Arnica montana L.: effects of mycorrhizal fungi and competing plants

Arnica montana is a rare plant that needs special protection because of its intensive harvesting for medicinal purposes. The present work was aimed at finding optimal culture conditions for Arnica plants in order to enable their successful reintroduction into their natural stands. Plants were cultivated under controlled greenhouse conditions on substrata with different nitrogen (N) concentration. As Arnica is always colonized by arbuscular mycorrhizal fungi (AMF) in nature, a fact that has been overlooked in other similar projects, we, here, applied and tested different inocula. We found that they differed in their effectiveness, both in establishing symbiosis, assessed by the colonization parameters, and in improving the performance of Arnica, evaluated by the photosynthetic parameters derived from the fluorescence transients (JIP-test), with the inocula containing G. intraradices or composed of several Glomus strains being the most effective. The comparison was possible only on substrata with medium N, since high N did not permit the formation of mycorrhiza, while at low N, few nonmycorrhizal plants survived until the measurements and mycorrhizal plants, which were well growing, exhibited a high heterogeneity. Analysis of secondary metabolites showed clearly that mycorrhization was associated with increased concentrations of phenolic acids in roots. For some of the inocula used, a tendency for increase of the level of phenolic acids in shoots and of sesquiterpene lactones, both in roots and in shoots, was also observed. We also studied the interactions between A. montana and Dactylis glomerata, known to compete with Arnica under field conditions. When specimens from both species were cultured together, there was no effect on D. glomerata, but Arnica could retain a photosynthetic performance that permitted survivability only in the presence of AMF; without AMF, the photosynthetic performance was lower, and the plants were eventually totally outcompeted.

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts.

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O2-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts.

On the question of the light-harvesting role of β-carotene in photosystem II and photosystem I core complexes.

β-Carotene is the only carotenoid present in the core complexes of Photosystems I and II. Its proximity to chlorophyll a molecules enables intermolecular electronic interactions, including β-carotene to chlorophyll a electronic excitation transfers. However, it has been well documented that, compared to chlorophylls and to phycobilins, the light harvesting efficiency of β-carotenes for photosynthetic O2 evolution is poor. This is more evident in cyanobacteria than in plants and algae because they lack accessory light harvesting pigments with absorptions that overlap the β-carotene absorption. In the present work we investigated the light harvesting role of β-carotenes in the cyanobacterium Synechococcus sp. PCC 7942 using selective β-carotene excitation and selective Photosystem detection of photo-induced electron transport to and from the intersystem plastoquinones (the plastoquinone pool). We report that, although selectively excited β-carotenes transfer electronic excitation to the chlorophyll a of both photosystems, they enable only the oxidation of the plastoquinone pool by Photosystem I but not its reduction by Photosystem II. This may suggest a light harvesting role for the β-carotenes of the Photosystem I core complex but not for those of the Photosystem II core complex. According to the present investigation, performed with whole cyanobacterial cells, the lower photosynthesis yields measured with β-Car-absorbed light can be attributed to the different excitation trapping efficiencies in the reaction centers of PSI and PSII.

Light and Heat Stress Adaptation of the Symbionts of Temperate and Coral Reef Foraminifers Probed in Hospite by the Chlorophyll a Fluorescence Kinetics

Since the early 80’s massive bleaching affects the reef ecosystem. It involves, besides corals, several other species among which large foraminifers, and it corresponds to the loss of their photosynthetic symbionts or the symbionts’ pigments. The cause is unclear, though temperature elevation and strong irradiation have been considered to be primary factors. In this work we investigated in two genera of coral reef foraminifers (Amphistegina lobifera and Amphisorus heimprichii) and in the temperate foraminifer Sorites variabilis the response of photosystem II (PSII) of their symbionts in hospite upon light stress (white light of 550 μE m-2 s-1 and red light of 3200 |μE m-2 s-1) and heat stress (up to 32 °C), by means of the Chla fluorescence transients O-J-I-P they exhibit upon illumination. The transients were analysed according to the JIP-test which leads to the calculation of several structural and functional parameters providing a quantification of PSII behaviour. We observed that the various parameters undergo modifications that differ concerning both their extent and their degree of elasticity, thus indicating that different adaptive strategies are employed in response to stress. The most pronounced of these regulatory changes is a wide decrease of the quantum yield of electron transport. However, the extent of the changes, different for the three studied species, was in general smaller when the cultures were kept under low light (70 μE m-2 s-1) than in darkness. By the applied stressors, PSII was not damaged and, except for some cells in which an expulsion of symbionts was initiated, no bleaching was observed. This can be well correlated with the observed adaptability of PSII. As a working hypothesis, it is proposed that the decrease of the capacity for electron transport activity might be among the factors triggering bleaching in the field

Experimental Resolution and Theoretical Complexity Determine the Amount of Information Extractable from the Chlorophyll Fluorescence Transient OJIP

Models Of Any Theoretical Complexity Level Can Be Formulated, But They Are Meaningful Only If They Can Be Experimentally Validated. We Focus On The Contribution Of Our Laboratory In Utilising Chlorophyll A Fluorescence Transients To Construct And Test Photosynthetic Models.

Role of Beneficial Microsymbionts on the Plant Performance and Plant Fitness

The number ofmicrobes in the rhizosphere of higher plants ismuch higher in comparison to bulk soil (Hiltner 1904). Due to this positive rhizosphereeffect, there are active and passive physical and chemical changes in the root system of the higher plants, which may have a great impact both on their nutrient status and their growth (Biro 2003). Among the microbes present in the rhizosphere, the microsymbiont bacteria and fungi are the most important for the plant growth and development. Their role and capacity for the biological nitrogen fixation and phosphorousmobilisation is quite established (Barea et al. 2002). The translocation of macroand micronutrients in these zones is influenced by the enhanced microbial activities. The effects aremediatedbydirect transfer of nutrients fromplant by the increased root system, and also by improving the competitiveness of higher plants in the nutrient uptake. Among the beneficial microbes, the associative and symbiotic N2 bacteria and the arbuscular mycorrhizal fungi (AMF) are the most common in the rhizosphere of higher plants. Artificial seed and soil inoculation techniques are used as a simple application of a mixed nodule extracts, or as a soil-root mixture (Hiltner 1904). However, the introduced microbes usually enter in competition with the native microflora in the soil (Graham 1992). The negative effects of the abiotic environmental stress factors (temperature, drought, acidity etc.) are also common (Graham 1992; Bayoumi et al. 1995). The final influence of any microbial inoculation in the rhizosphere, therefore, is the result of the complex interactions between the plants, the rhizosphere inhabitants and the different microbial and environmental components involved (Postma et al. 1989). The antagonistic and synergistic behaviours, of the beneficial microsymbionts is a crucial step considering the plant growth and their sustainability (Hoflich 1993).

Activity and Heterogeneity of PSII Probed in Vivo by the Chlorophyll a Fluorescence Rise O-(K)-J-I-P

The photosynthetic organisms undergo in nature perpetual state changes in order to adapt to a perpetually changing environment (1). The environmental changes are provoked by many different factors, natural and physical/chemical, as well as any combination of any of them. Therefore, and moreover considering the huge heterogeneity of the photosynthetic material in nature, a plethora of survival strategies is employed. However, a survival strategy is a combination of adaptive processes, each regulating structural features which consequently determine function (1). Therefore, the plethora of macroscopically different strategies might rather arise from many possible combinations of much fewer adaptive processes. A certain combination may be specific for a certain behavioural type.