JIP Test Research Articles

Effect of water deficit and potassium fertilization on photosynthetic activity in cotton plants

Physiological mechanisms that can contribute to drought tolerance and the role of potassium fertilization in cotton were studied by evaluation of parameters describing photosynthetic performance. Gas-exchange and chlorophyll fluorescence characteristics were measured on leaves of two cotton genotypes, one drought sensitive (Nazilli 84-S) and the other drought tolerant (Sahin 2000), grown in field conditions in the Aegean region of Turkey under different regimes of water and potassium supply. It was shown that under drought conditions without potassium fertilization Sahin 2000 had a higher photosynthetic rate and stomatal conductance than Nazilli 84-S. Potassium fertilization to a great extent compensated for the inhibitory effect of drought on photosynthesis. Application of the JIP test by using chlorophyll fluorescence data revealed that the drought sensitive Nazilli 84-S was more responsive to potassium fertilization than Sahin 2000, as judged by a number of parameters representing quantum efficiency of the processes and energy fluxes in photosystem (PS) II. The observed decrease in photosynthetic CO2 assimilation in both cotton cultivars under drought conditions was not accompanied by any significant decrease in the electron transport flux in PSII and maximum quantum yield of primary photochemistry.

The use of JIP test to evaluate drought-tolerance of transgenic rice overexpressing OsNAC10

In this study, the JIP test was exploited to assess drought-tolerance of transgenic rice overexpressing OsNAC10. Two types of promoters, RCc3 (root-specific) and GOS2 (constitutive), were used to drive the transcription factor OsNAC10, a gene involved in diverse functions including stress responses. Three-month-old plants were exposed to drought for 1 week and their fluorescence kinetics was evaluated. Our results showed that drought-treated non-transgenic plants (NT) have higher fluorescence intensity at the J phase (2 ms) compared to transgenic plants, indicating a decline in electron transport beyond the reduced plastoquinone (QA −). As manifested by negative L bands, transgenic plants also showed higher energetic connectivity and stability over NT plants under drought conditions. Also, the pool size of the end electron acceptor at the photosystem I was reduced more in NT than in transgenic plants under drought conditions. Furthermore, the transgenic plants had higher PItotal, a combined parameter that reflects all the driving forces considered in JIP test, than NT plants under drought conditions. In particular, the PItotal of the RCc3:OsNAC10 plants was higher than that of NT plants, which was in good agreement with their differences in grain yield. Thus, the JIP test proved to be practical for evaluating drought-tolerance of transgenic plants.

Inherent nitrogen deficiency in Pistacia lentiscus preferentially affects photosystem I: a seasonal field study.

Although it is widely documented that CO2 assimilation rates are positively correlated with leaf nitrogen, corresponding studies on a link between this nutrient and photosynthetic light reactions are scarce, especially under natural field conditions. In this investigation, we exploited natural variation in the nitrogen content of mature leaves of Pistacia lentiscus L. (mastic tree) in conjunction with fast chlorophyll a fluorescence rise (the OJIP curves) analysed according to the 'JIP test', as this was recently modified to allow for the assessment of events in or around PSI. The results depended on the sampling season, with low nitrogen leaves displaying lower efficiencies for electron flow from intermediate carriers to final PSI acceptors, and lower relative pool sizes of these acceptors, both during the autumn and winter. However, parameters related to the PSII) activity (i.e. quantum yields for photon trapping and electron flow along PSII and the efficiency of a trapped exciton to move an electron from the first plastoquoinone electron acceptor of PSII to intermediate carriers) were limited by low nitrogen only during the winter period. As a result, parameters like the quantum yield of total electron flow along both photosystems as well as the total photosynthetic performance index (PItotal) were positively correlated with leaf nitrogen independently of the season. We conclude that nitrogen deficiency under field conditions preferentially affects PSI activity while the effects on PSII are evident only during the stressful period of the year.

Revegetation of a Uranium Mine Dump by Using Fertilizer Treated Sessile Oaks

The rehabilitation of contaminated sites and the establishment of suitable trees for revegetation purposes is often problematic due to the mostly suboptimal nutrient supply and the poor humus reservoir. For these reasons hydrogels (Stockosorb®) and novel humus substitutes (NOVIHUM®), serving as long lasting fertilizer (LLF), were recently tested successfully. At the beginning of this multiyear study, those LLFs were administered to the root zone of young sessile oaks (Quercus petraea (Mattuschka) Liebl.), growing in test trials on a uranium mine dump in Schlema (Germany). To quantify the effect of LLFs on plant vitality, chlorophyll a fluorescence measurements and JIP test analyses were used. The results revealed up to 49% higher average photosynthetic vitality (PIABS) of the LLF treated plants compared to controls. Particularly in the first test year, the efficiency of photosynthetic electron transport was strongly increased. This stimulation of photosynthetic activity was supported by direct measurements showing up to 129% increased diameter growth of the treated plants after a four year experimental period.Furthermore an increase of the maximum water holding capacity of the dump soil was attained by using LLFs. Overall, the findings reported here represent a feasible, ecologically justifiable reforestation method with a low environmental hazard potential.

Analysis of high temperature stress on the dynamics of antenna size and reducing side heterogeneity of Photosystem II in wheat leaves ( Triticum aestivum)

This study demonstrates the effect of high temperature stress on the heterogeneous behavior of PSII in Wheat ( Triticum aestivum) leaves. Photosystem II in green plant chloroplasts displays heterogeneity both in the composition of its light harvesting antenna i.e. on the basis of antenna size (α, β and γ centers) and in the ability to reduce the plastoquinone pool i.e. the reducing side of the reaction centers (Q B-reducing centers and Q B-non-reducing centers). Detached wheat leaves were subjected to high temperature stress of 35 °C, 40 °C and 45 °C. The chlorophyll a (Chl a) fluorescence transient were recorded in vivo with high time resolution and analyzed according to JIP test which can quantify PS II behavior using Plant efficiency analyzer (PEA). Other than PEA, Biolyzer HP-3 software was used to evaluate different types of heterogeneity in wheat leaves. The results revealed that at high temperature, there was a change in the relative amounts of PSII α, β and γ centers. As judged from the complementary area growth curve, it seemed that with increasing temperature the PSII β and PSII γ centers increased at the expense of PSII α centers. The reducing side heterogeneity was also affected as shown by an increase in the number of Q B-non-reducing centers at high temperatures. The reversibility of high temperature induced damage on PSII heterogeneity was also studied. Antenna size heterogeneity was recovered fully up to 40 °C while reducing side heterogeneity showed partial recovery at 40 °C. An irreversible damage to both the types of heterogeneity was observed at 45 °C. The work is a significant contribution to understand the basic mechanism involved in the adaptation of crop plants to stress conditions.

Characterization of photosystem II heterogeneity in response to high salt stress in wheat leaves (Triticum aestivum)

The effect of high salt stress on PS II heterogeneity was investigated in wheat (Triticum aestivum) leaves. On the basis of antenna size, PS II has been classified into three forms, i.e., α, β, and γ centers while on the basis of electron transport properties of the reducing side of the reaction centers, two distinct forms of PS II have been suggested, i.e., Q(B) reducing centers and Q(B) non-reducing centers. The chlorophyll a (Chl a) fluorescence transients, which can quantify PS II behavior, were recorded using PEA to derive OJIP in vivo with high time resolution and further analyzed according to JIP test. Our results showed that with an increase in the salt concentration during growth, the number of Q(B) non-reducing centers increased. In antenna size heterogeneity the number of β and γ centers increased while the number of α centers decreased. A change in the energetic connectivity between the PS II units was also observed. Recovery studies showed that antenna heterogeneity was completely recovered from damage at 0.5M NaCl concentration and partially recovered at 1M NaCl concentration while reducing side heterogeneity showed no recovery at all after 0.5M onwards.

The JIP test: a tool to screen the capacity of plant adaptation to climate change

Adaptation and acclimation are strategies that forests enact to cope with climate change. They consist of genotypic and phenotypic adjustments that allow plants to grow and reproduce successfully in a stressful environment. Both these aspects can be actively promoted by reforestation programmes. The key phases are: (1) selection of the most suitable provenances and genotypes; (2) adoption of adequate cultural techniques in nursery; and (3) monitoring of the plantations. Physiological techniques may be useful to assist all the phases of this process. Among these, chlorophyll fluorescence-based techniques, such as the JIP test, are relevant to monitor the stress conditions and the effectiveness of the cultural practices. JIP test is a non-destructive, non-invasive, informative, very fast and inexpensive technique, and can be used to support the cultural decision.

Salt stress induces a decrease in excitation energy transfer from phycobilisomes to photosystem II but an increase to photosystem I in the cyanobacterium Spirulina platensis

The effects of salt stress (0-0.8M NaCl) on excitation energy transfer from phycobilisomes to photosystem I (PSI) and photosystem II (PSII) in the cyanobacterium Spirulina platensis were investigated. Salt stress resulted in a significant decrease in photosynthetic oxygen evolution activity and PSII electron transport activity, but a significant increase in PSI electron transport activity. Analyses of the polyphasic fluorescence transients (OJIP) showed that, with an increase in salt concentration, the fluorescence yield at the phases J, I and P declined considerably and the transient almost leveled off at 0.8M NaCl. Analyses of the JIP test demonstrated that salt stress led to a decrease in the maximal efficiency of PSII photochemistry, the probability of electron transfer beyond Q(A), and the yield of electron transport beyond Q(A). In addition, salt stress resulted in a decrease in the electron transport per PSII reaction center, but an increase in the absorption per PSII reaction center. However, there was no significant change in the trapping per PSII reaction center. Furthermore, there was a decrease in the concentration of the active PSII reaction centers. Analyses of 77K chlorophyll fluorescence emission spectra excited either at 436 or 580nm showed that salt stress inhibited excitation energy transfer from phycobilisomes to PSII but induced an increase in the efficiency of energy transfer from phycobilisomes to PSI. Based on these results, it is suggested that, through a down-regulation of PSII reaction centers and a shift of excitation energy transfer in favor of PSI, the PSII apparatus was protected from excess excitation energy.

Arbuscular mycorrhizal fungi alter thymol derivative contents of Inula ensifolia L.

Individuals of Inula ensifolia L. (Asteraceae), a valuable xerothermic plant species with potential therapeutic value, were inoculated under laboratory conditions with different strains of arbuscular mycorrhizal fungi (AMF): (1) Glomus intraradices UNIJAG PL-Bot, (2) G. intraradices UNIJAG PL-Kap, (3) Glomus clarum UNIJAG PL13-2, and (4) AMF crude inoculum from natural stands of I. ensifolia. We found AMF species specificity in the stimulation of thymol derivative production in the roots of I. ensifolia. There was an increase in thymol derivative contents in roots after G. clarum inoculation and at the same time the decreased production of these metabolites in the G. intraradices treatments. Moreover, no correlation between the extent of AMF colonization and the effects of the fungal symbionts on the plant was observed. A multilevel analysis of chlorophyll a fluorescence transients (JIP test) permitted an evaluation of plant vitality, expressed in photosynthetic performance index, influenced by the applied AMF strains, which was found to be in good agreement with the results concerning thymol derivative production. The mechanisms by which AMF trigger changes in phytochemical concentration in plant tissues and their consequences for practice are discussed.

BORON DEFICIENCY AFFECTS GAS EXCHANGE AND PHOTOCHEMICAL EFFICIENCY (JPI TEST PARAMETERS) IN GREEN DWARF COCONUT

Boron (B) deficiency causes a wide array of symptoms, not only among species of palms, but also within a single species (i.e. Cocos nucifera). A better understanding of the effects of B deficiency in coconut will be important to try optimizing a rational fertilization management in coconut plants. Thus, modification of PSII photochemistry (using a group of fluorescence parameters, called the JIP- test, that quantify the stepwise flow of energy through Photosystem II) and gas-exchange in boron deficient green dwarf coconut plants were investigated. Our results suggest that a modification of PSII photochemistry (non-stomatic effects) and gas-exchange (stomatic effects) were induced by boron deficiency. Such modifications are manifested by (1) increase the ratio of total dissipation to the amount of active reaction centers (RCs) [dissipation (DI)/RC] and (2) leaf-to-air vapor pressure difference (VPDleaf-air). These modifications (on PSII photochemistry and gas-exchange) were caused by a decrease in energy absorbed per excited cross-section [absorption flux (ABS)/cross section of the sample (CS0)], density of active reaction centers (RC/CS), maximal trapping rate of an exciton that will lead to QA reduction measured over a cross- section of active and inactive RCs [trapping flux (TR)/CS0], electron transport per excited cross-section [electron transport flux (ET0)/CS)], area above curve (proportional to the pool size of the electron acceptors QA on the reducing side of PSII), photosynthesis (A), stomatal conductance (gs), transpiration (E), chlorophyll concentration (SPAD readings), growth parameters (root DW and height plant). Our results demonstrate that by analyzing fluorescence (JIP test parameters) derived from the polyphasic fluorescence transients measurements were able to estimate the functional changes of PSII in B deficient coconut plants. The results in this study suggest that fluorescence analysis (JIP test) and instantaneous measurements of gas-exchange can be useful tools in assessing the physiological effects of B deficiency in green dwarf coconut.

EFFECT OF CHROMIUM(VI) ON PHOTOSYSTEM II ACTIVITY AND HETEROGENEITY OF SYNECHOCYSTIS SP. (CYANOPHYTA): STUDIED WITH IN VIVO CHLOROPHYLL FLUORESCENCE TESTS(1).

The inhibitory effect of Cr(VI) on the PSII of Synechocystis sp. was studied. Cr(VI) reduced O2 evolution and inhibited the water-splitting system in PSII. S-states test and flash induction test showed that Cr(VI) exposure increased the proportion of inactivated PSII (PSIIX ) and PSIIβ reaction centers, which increased the fluxes of dissipated energy. JIP test and QA (-) reoxidation test demonstrated that Cr(VI) treatment induces inhibition of electron transport from QA (-) to QB /QB (-) and accumulation of P680 (+) . More QA (-) had to be oxidized through S2 (QA QB )(-) charge recombination and oxidation by PQ9 molecules in PSII under Cr(VI) stress. These changes finally decreased the index of photosynthesis performance.

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.

Influence of CO2 concentrating mechanism on photoinhibition in Synechococcus sp. PCC7942 (Cyanophyceae)

H. Cheng, G. Dai, L. Yu, X. Zhong, P. Juneau and B. Qiu. 2008. Influence of CO2 concentrating mechanism on photoinhibition in Synechococcus sp. PCC7942 (Cyanophyceae). Phycologia 47: 588–598. DOI: 10.2216/07-44.1.In this study the effects of inorganic carbon transport in the cyanobacterium Synechococcus sp. PCC7942 on its photoinhibition were investigated. The photosynthetic oxygen evolution of samples under a strong irradiance treatment at 100 µM KHCO3 was much higher than that at 10,000 µM KHCO3. After strong irradiance treatment, the abundance of the sbtA transcript encoding a Na+-dependent transporter was significantly higher under treatment at 100 µM KHCO3 than at 10,000 µM KHCO3. Lincomycin had little effect on the difference of photosystem II (PSII) activity decline under these two treatments, which suggests that CCM activity reduces photodamage but does not boost the rate of PSII repair. The maximal photosynthetic efficiency was decreased significantly during the strong irradiance treatment, and its decrease was enhanced when the external DIC concentration was raised from 100 to 10,000 µM KHCO3. The JIP test showed that there were more active reaction centres under low Ci conditions after high light treatment. Studies on 77-K fluorescence emission spectra showed that the fluorescence peaks F692 nm and F710 nm in 10,000-µM KHCO3 treated samples both decreased after strong irradiance treatment and were significantly smaller than those of 100-µM KHCO3 treated samples. Samples treated with 100 µM KHCO3, compared to 10,000 µM KHCO3 had a higher possibility of transmitting an exciton to a closed reaction centre rather than to an open reaction centre and thus to reduce the overexcitation of the reaction centres. These results indicated that higher external inorganic carbon concentration was associated with more severe photoinhibition under high irradiance and that the operation of CO2 concentrating mechanism might reduce the overexcitation of reaction centres to reduce photodamage.

Effect of dibromothymoquinone on Chlorophyll a Fluorescence in Chlamydomonas reinhardtii cells incubated in complete or sulfur-depleted medium

The influence of dibromothymoquinone on chlorophyll fluorescence was studied in Chlamydomonas reinhardtii cells using PAM and PEA fluorometers. The reagent affected differently control cells incubated in complete medium and S-starved cells. Thus, the fluorescence yield in the control essentially increased in the presence of dibromothymoquinone, which can be due to the inactivation of light-harvesting complex II protein kinase, followed by the suppression of membrane transition from high-fluorescence state 1 to low-fluorescence state 2. On the contrary, S-starved cells with membranes in state 2 showed a lower fluorescence yield in the presence of dibromothymoquinone than without it. The JIP test of OJIP fluorescence transients suggests that dibromothymoquinone inhibits both light-harvesting complex II kinase and photosynthetic electron transport when added to control, while in starved cells, it acts predominantly as an electron acceptor.

Response of plants to ectomycorrhizae in N-limited conditions: which factors determine its variation?

In the present work, the following hypotheses were tested: (1) the negative effects of mycorrhization over host plant productivity in N-limited conditions are due to N retention by the fungal partner and not due to excessive C drainage; (2) If mycorrhization results in decreased N uptake, the host plant decreases its C investment in fungal growth. The effects of mycorrhization over a wide range of combinations between N availability, N concentration in plant tissues, and degree of mycorrhizal colonization were studied in Pinus pinaster L. mycorrhizal with Pisolithus tinctorius. Several plant productivity parameters, the seedlings' N status, chl a fluorescence (JIP test), and mycorrhizal colonization were measured. N was always limiting. A gradient of mycorrhizal effects over the host plant's growth and vitality was successfully obtained. The mycorrhizal effects on plant growth and N uptake were very strongly and positively correlated, and no evidence was found of a C limitation to growth, confirming hypothesis 1. Indications were found that the plants continued to provide C to the fungus although the N supplied by it was increasingly lower, denying hypothesis 2. A new index, the mycorrhizal N demand-supply balance, was found to efficiently explain, and to have a curvilinear relation with, the variation in response to mycorrhization. The mycorrhizal effect on host plant growth was not related to a negative effect on its photosynthetic performance and, therefore, reflected changes in resource allocation between host plant and mycorrhizal fungus, not in plant vitality.

JIP test for fast fluorescence transients as a rapid and sensitive technique in assessing the effectiveness of arbuscular mycorrhizal fungi in Zea mays: Analysis of chlorophyll a fluorescence

We applied chlorophyll a fluorescence as a biomarker to assess the growth enhancement of maize plants after inoculation with the mycorrhizal fungi Piriformospora indica, Glomus mosseae, or a mixed culture (P. indica, G. mosseae and G. caledonium), and compared it with uninoculated plants (controls). The quantum efficiencies for pigment system II (ϕ Po) were not much affected. The yield for electron flow (ϕ Eo = ϕ Po *ψo) was highly responsive to P. indica and mixed culture. Electron transport per trapping center (ψo) was remarkably influenced by the endophytes. Plants treated with P. indica and mixed culture showed relatively higher quantum yield compared to uninoculated plants. Absorption per reaction center was influenced by the endophytes. Piriformospora indica followed by mixed inoculum showed slightly lower values as compared to controls. The morphological markers, namley height of the plants and percentage colonization in the roots, reflected the biophysiological markers (chlorophyll a fluorescence); hence, the JIP test (a test applied to analyze fast fluorescence kinetics O-J-I-P) can be used as a sensitive and rapid tool for determining the efficiency of mycorrhizal fungi in host plants.

Study of photosystem 2 heterogeneity in the sulfur-deficient green alga Chlamydomonas reinhardtii

A set of chlorophyll fluorescence methods, including PEA (Plant Efficiency Analyser), PAM (Pulse Amplitude Modulated fluorometer), and picosecond fluorometer, was employed to study PS 2 heterogeneity in sulfur deprived green algae Chlamydomonas reinhardtii. The regression method and JIP test were applied to analyze chlorophyll fluorescence kinetics. The fractions of PS 2 characterized by the energetic disconnection, smaller antenna size, elevated constant rate of primary photochemistry, and inability to maintain DeltapH-dependent energy dissipation increased essentially already after 12 h of incubation in sulfur depleted medium. The amount of PS 2 centers with reduced QA (closed state), QB-non-reducing centers with impaired water splitting function, and centers coupled to the plastoquinone pool with the slow cycle rate increased dramatically after 24 h period of deprivation. The mechanisms of PS 2 inactivation under sulfur deprivation are discussed.

The OJIP fast fluorescence rise characterizes Graptophyllum species and their stress responses

Causes for rarity in plants are poorly understood. Graptophyllum reticulatum is an endangered endemic species, and it has three close relatives with different conservation status: the vulnerable G. ilicifolium, the rare G. excelsum, and the common G. spinigerum. Applied to the chlorophyll a fluorescence transient of leaves, the JIP test provides a Performance Index (PI) which quantifies the main steps in photosystem II (PSII) photochemistry including light energy absorption, excitation energy trapping, and conversion of excitation energy into electron flow. The PI is calculated from three components which depend on the reaction center density, the trapping efficiency, and the electron transport efficiency. PI was measured in the natural habitats of the four species and under artificially imposed environmental stresses in the glasshouse to determine whether conservation status was related to stress resilience. The results showed that soil type is unlikely to restrict the endangered G. reticulatum, vulnerable G. ilicifolium, or rare G. excelsum because PI was similar in plants grown in diverse soils in the glasshouse. Photoinhibition is likely to restrict the endangered G. reticulatum to shade habitats because PI was significantly reduced when plants were exposed to more than 15% ambient light in controlled experiments. Water availability may determine the location and distribution of the vulnerable G. ilicifolium and common G. spinigerum because PI was reduced more than 60% when plants were exposed to water stress. While the characteristics of their natural habitats correspond to and explain the physiological responses, there was no obvious relationship between conservation status and environmental resilience. PI can be used to monitor vigor and health of populations of plants in the natural habitat. In cultivation experiments PI responds to key environmental variables that affect the distribution of species with conservation significance.

Alternaria alternataCrofton-Weed Toxin: a Natural Inhibitor of Photosystem II inChlamydomonas reinhardtiiThylakoids

The action site of Alternaria alternata Crofton-weed toxin (AAC-toxin), isolated first from Alternaria alternata (Fr.) Keissler, was investigated in Chlamydomonas reinhardtii thylakoids. The results revealed that AAC-toxin inhibited photophosphorylation in a concentration-dependent pattern. Similarly, toxin inhibited uncoupled, basal electron flow and photosystem II (PSII) electron transport as well. However, toxin did not affect photosystem I (PSI) activity or the partial reaction of electron transport from H2O to silicomolybdic acid (SiMo). Therefore, the action site of toxin was located at QB level. In addition, the toxin may behave as an energy-transfer inhibitor at high concentrations by inhibiting phosphorylating electron transport and Mg2+ATPase activity. Chlorophyll a fluorescence induction and JIP test corroborated the inhibition at QB level. Through observations of the different sensitivity of toxin on D1 mutants of C. reinhardtii, evidence further confirmed that AAC-toxin inhibited electron transport by displacing the QB on the D1 protein, and the mode of action was similar to phenol-type PSII inhibitors.

Photosynthesis responses to ozone in young trees of three species with different sensitivities, in a 2‐year open‐top chamber experiment (Curno, Italy)

This paper reports the findings of an open‐top chamber experiment carried out in northern Italy (Forest nursery at Curno), during the 2004 and 2005 growth seasons, on Fagus sylvatica and Quercus robur seedlings and on Populus nigra cuttings, in order to test their photosynthesis response to ambient ozone. The experimental protocols were non‐filtered air (NF), charcoal‐filtered air (CF) and open air (OA). Tests performed included morphological features of leaves; development of foliar symptoms; chlorophyll content, determined by non‐destructive means; chlorophyll fluorescence (direct fluorescence and JIP test) and gas exchanges and net photosynthesis (PN). Main findings were as follows: (1) symptoms occurred early and were extensive in P. nigra, and they occurred later in F. sylvatica, whereas early degeneration of chlorophyll occurred in late summer in Q. robur; (2) in conditions of ozone exposure, the three species all presented a decline in photosynthesis efficiency and a decrease in PN, regardless of the symptomatology they displayed; (3) leaf traits are predictors of species‐specific sensitivity to ozone—the high density of Q. robur foliar tissues prevents this species from developing visible symptoms and reduces the extent of physiological responses and (4) physiological responses varied from year to year in the same species—responses were lower in the second year of the experiment, when plants had become better acclimatized to plot conditions.