Evaluation of chlorophyll fluorescence as a tool for salt stress detection in roses

The induction kinetic of the chlorophyll (Chl) fluorescence and the F_v/F_m ratio have been tested in order to find out the suitability of this technique to evaluate damage caused by salinity in plants of Rosa hybrida cv. Ilseta grafted on R. manetti growing in a greenhouse under non-saturating irradiance. Under these conditions salinity induced changes in plants morphology, nutrient and Chl contents and in the gas exchange parameters, but not in the F_v/F_m ratio. The Rfd index did not reveal more information. The F_v/F_m ratio as well as the fluorescence induction curves were more affected by salinity when an irradiation stress was added, therefore as an indicator of salt stress in roses, Chl fluorescence is of limited use when the plants are grown without additional stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Changes in yield ofin-vivo fluorescence of chlorophyll a as a tool for selective herbicide monitoring

Triazines and derivatives of phenylurea, which are often found in outdoor water samples, induce specific changes in the yield of thein-vivo chlorophyll -fluorescence of PSII. These changes are correlated quantitatively with the concentration of the herbicides and can therefore be used to set-up a low-price monitor system. In order to detect selectively the herbicide-sensitive part of the fluorescence emission a pulse amplitude modulated fluorimeter was used. The bioassay system was optimised with respect to test organism, growing and measuring conditions. The relationship between fluorescence yield and herbicide concentrations were experimentally determined for the triazines atrazine and simazine and the phenylurea herbicide DCMU and mathematically fitted (r=0.99). The I₅₀-values were 0.9 µM for DCMU, 2.2 µM for simazine and 3.3 µM for atrazine. The detection limit of about 0.5 µM clearly shows that the sensitivity of this bioassay system is too low to reach the requirements of the drinking water regulation. However, due to its insensitivity against complex water matrices, there is good hope to combine this fluorometric bioassay with a potent herbicide preconcentration method like a solid-phase extraction procedure.Author for correspondence     

Chlorophyll fluorescence as a tool for nutrient status identification in rapeseed plants

In natural conditions, plants growth and development depends on environmental conditions, including the availability of micro- and macroelements in the soil. Nutrient status should thus be examined not by establishing the effects of single nutrient deficiencies on the physiological state of the plant but by combinations of them. Differences in the nutrient content significantly affect the photochemical process of photosynthesis therefore playing a crucial role in plants growth and development. In this work, an attempt was made to find a connection between element content in (i) different soils, (ii) plant leaves, grown on these soils and (iii) changes in selected chlorophyll a fluorescence parameters, in order to find a method for early detection of plant stress resulting from the combination of nutrient status in natural conditions. To achieve this goal, a mathematical procedure was used which combines principal component analysis (a tool for the reduction of data complexity), hierarchical k-means (a classification method) and a machine-learning method—super-organising maps. Differences in the mineral content of soil and plant leaves resulted in functional changes in the photosynthetic machinery that can be measured by chlorophyll a fluorescent signals. Five groups of patterns in the chlorophyll fluorescent parameters were established: the ‘no deficiency’, Fe-specific deficiency, slight, moderate and strong deficiency. Unfavourable development in groups with nutrient deficiency of any kind was reflected by a strong increase in F _o and ΔV/Δt ₀ and decline in φ _Po, φ _Eo δ _Ro and φ _Ro. The strong deficiency group showed the suboptimal development of the photosynthetic machinery, which affects both PSII and PSI. The nutrient-deficient groups also differed in antenna complex organisation. Thus, our work suggests that the chlorophyll fluorescent method combined with machine-learning methods can be highly informative and in some cases, it can replace much more expensive and time-consuming procedures such as chemometric analyses.     

Delayed fluorescence as a universal tool for the measurement of circadian rhythms in higher plants

The plant circadian clock plays an important role in enhancing performance and increasing vegetative yield. Much of our current understanding of the mechanism and function of the plant clock has come from the development of Arabidopsis thaliana as a model circadian organism. Key to this rapid progress has been the development of robust circadian markers, specifically circadian-regulated luciferase reporter genes. Studies of the clock in crop species and non-model organisms are currently hindered by the absence of a simple high-throughput universal assay for clock function, accuracy and robustness. Delayed fluorescence (DF) is a fundamental process occurring in all photosynthetic organisms. It is luminescence-produced post-illumination due to charge recombination in photosystem II (PSII) leading to excitation of P680 and the subsequent emission of a photon. Here we report that the amount of DF oscillates with an approximately 24-h period and is under the control of the circadian clock in a diverse selection of plants. Thus, DF provides a simple clock output that may allow the clock to be assayed in vivo in any photosynthetic organism. Furthermore, our data provide direct evidence that the nucleus-encoded, three-loop circadian oscillator underlies rhythms of PSII activity in the chloroplast. This simple, high-throughput and non-transgenic assay could be integrated into crop breeding programmes, the assay allows the selection of plants that have robust and accurate clocks, and possibly enhanced performance and vegetative yield. This assay could also be used to characterize rapidly the role and function of any novel Arabidopsis circadian mutant.     

Growth, photosynthesis and chlorophyll fluorescence of sweet pepper plants as affected by the cultivation method

Different methods of sweet pepper cultivation (organic, integrated and conventional farming) were evaluated under greenhouse conditions. Organic treatment was defined as only 4‐kg manure application at the preplanting stage. Conventional and integrated treatments consisted of application of the manure dosage plus chemical fertiliser at 100% and 50% of the local farmers’ amount, respectively, to avoid nutrient depletion. Plant growth parameters such as shoot dry matter, total leaf area and leaf weight fraction were all reduced in the organic treatment compared with the conventional. Leaf expansion was dramatically reduced in the organic treatment 155 days after transplanting. Relative growth rates were significantly affected by the nitrogen concentration in each organ and were directly related to the cultivation method. Chlorophylls (a + b) contents in the leaves were reduced in the organic treatment and were directly correlated with the nondestructive quantification of chlorophylls using a portable chlorophyll meter. Net photosynthesis was also reduced in the organic treatment, but chlorophyll fluorescence was not affected. This study shows that biometric monitoring and fast and non‐invasive techniques of plant nutrient status analysis could help to improve growth as easy and useful tools to follow the nutrient status at different phenological stages, especially when no chemical fertilisers are applied.     

Tracking viral movement in plants by means of chlorophyll fluorescence imaging

Many techniques have been applied to understand viral cell-to-cell movement in host plants, but little progress has been made in understanding viral vascular transport mechanisms. We propose the use of chlorophyll fluorescence imaging techniques, not only to diagnose the viral infection, but also to follow the movement of the virus through the vascular system and its subsequent spread into the leaves. In Nicotiana benthamiana plants, imaging of chlorophyll fluorescence parameters such as Ф_PSII and NPQ proved useful to follow infections with Pepper mild mottle virus. The results demonstrate a correlation between changes in the chlorophyll fluorescence parameters and the viral distribution analyzed by tissue printing.     

Salt tolerance in crop plants monitored by chlorophyll fluorescence in vivo

The potential of measurements of chlorophyll fluorescence in vivo to detect cellular responses to salinity and degrees of salt stress in leaves was investigated for three crop plants. Sugar beet (Beta vulgaris L.) (salt tolerant), sunflower (Helianthus annuus L.) (moderately salt tolerant), and bean (Phaseolus Vulgaris L. cv Canadian Wonder) (salt intolerant) were grown in pots and watered with mineral nutrient solution containing 100 millimolar NaCl. The fast rise in variable chlorophyll fluorescence yield that is correlated with photoreduction of photosystem II acceptors increased in leaves of sugar beet plants treated with salt suggesting stimulation of photosystem II activity relative to photosystem I. In sunflower, this fast rise was depressed by approximately 25% and the subsequent slow rate of quenching of the chlorophyll fluorescence was stimulated. These differences were more marked in the older mature leaves indicating an increasing gradient of salt response down the plant. The salt effect in vivo was reversible since chloroplasts isolated from mature leaves of salt-treated and control sunflower plants gave similar photosystem II activities. Unlike in sugar beet and sunflower, leaves of salt-treated bean progressively lost chlorophyll. The rate of slow quenching of chlorophyll fluorescence decreased indicating development of a partial block after photosystem II and possible initial stimulation of photosystem II activity. With further loss of chlorophyll photosystem II activity declined. It was concluded that measurements of chlorophyll fluorescence in vivo can provide a rapid means of detecting salt stress in leaves, including instances where photosynthesis is reduced in the absence of visible symptoms. The possible application to screening for salt tolerance is discussed.     

Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

Mechanisms of non-photochemical chlorophyll fluorescence quenching in higher plants

The excitation energy of pigment molecules in photosynthetic antennae systems is utilised by photochemistry, partly it is thermally dissipated, and partly it is emitted as fluorescence. Changes in the quantum yield of chlorophyll (Chl) fluorescence reflect the changes in quantum yield of photochemical reaction and thermal dissipation of the excitation energy. Decrease of the Chl fluorescence quantum yield is called the Chl fluorescence quenching. The decrease of the quantum yield that is accompanied by photochemical reactions has been termed the photochemical quenching, and the decrease accompanied by thermal dissipation of the excitation energy is called the non-photochemical quenching. This review deals with mechanisms of the non-photochemical quenching.     

Conventional and combinatorial chlorophyll fluorescence imaging of tobamovirus-infected plants

We compared by chlorophyll (Chl) fluorescence imaging the effects of two strains of the same virus (Italian and Spanish strains of the Pepper mild mottle virus — PMMoV-I and-S, respectively) in the host plant Nicotiana benthamiana. The infection was visualized either using conventional Chl fluorescence parameters or by an advanced statistical approach, yielding a combinatorial set of images that enhances the contrast between control and PMMoV-infected plants in the early infection steps. Among the conventional Chl fluorescence parameters, the non-photochemical quenching parameter NPQ was found to be an effective PMMoV infection reporter in asymptomatic leaves of N. benthamiana, detecting an intermediate infection phase. The combinatorial imaging revealed the infection earlier than any of the standard Chl fluorescence parameters, detecting the PMMoV-S infection as soon as 4 d post-inoculation (dpi), and PMMoV-I infection at 6 dpi; the delay correlates with the lower virulence of the last viral strain.     

Analysis of temperature-jump chlorophyll fluorescence induction in plants.

A newly observed general chlorophyll fluorescence induction effect in plants is described. Fluorescence yield can rise through as many as four different phases (alpha, beta, gamma, ) in the dark, when intact cells or leaves are rapidly heated (within approx. 2.5 s) from 20 to 40-50 degrees C. An analysis of this temperature-jump fluorescence induction in Scenedesmus obliquus leads to the following: 1. Phase alpha is due to removal of S-quenching and appears to be related to heat deactivation of the water-splitting enzyme system. With prolonged heating, irreversibility of alpha upon recooling reflects irreversible damage to the water-splitting enzyme system. 2. beta is independent of the S-states and of the redox state of primary System II acceptor Q. It is suggested that beta parallels functional separation of Q from the System II trapping centre. This effect is highly reversible. 3. gamma and beta reflect reduction of primary System II acceptor Q by a heat-induced endogenous reductant, which is probably identical to hydrogenase. Critical temperatures for pronounced alpha and beta phases differ markedly in different plants. Possible correlations between temperature-jump fluorescence inductio, thylakoid membrane lipid composition, lipid phase transition and lipid-protein interactions are discussed.     

Skin fluorescence as a clinical tool for non-invasive assessment of advanced glycation and long-term complications of diabetes

Glycation is important in the development of complications of diabetes mellitus and may have a central role in the well-described glycaemic memory effect in developing these complications. Skin fluorescence has emerged over the last decade as a non-invasive method for assessing accumulation of advanced glycation endproducts. Skin fluorescence is independently related to micro- and macrovascular complications in both type 1 and type 2 diabetes mellitus and is associated with mortality in type 2 diabetes. The relation between skin fluorescence and cardiovascular disease also extends to other conditions with increased tissue AGE levels, such as renal failure. Besides cardiovascular complications, skin fluorescence has been associated, more recently, with other prevalent conditions in diabetes, such as brain atrophy and depression. Furthermore, skin fluorescence is related to past long-term glycaemic control and clinical markers of cardiovascular disease. This review will discuss the technique of skin fluorescence, its validation as a marker of tissue AGE accumulation, and its use as a clinical tool for the prediction of long-term complications in diabetes mellitus.     

Dimethoate-induced slow S to M chlorophyll a fluorescence transient in wheat plants

In eukaryotic oxygenic photosynthetic organisms (both plants and algae), the maximum fluorescence is at peak P, with peak M lying much lower, or being even absent. Thus, the PSMT phase, where S is semisteady state, and T is terminal state, is replaced by a monotonous P→T fluorescence decay. In the present study, we found that dimethoate-treated wheat plant leaves showed SM transient, whereas in the case of control plants monotonous P→T fluorescence decay occured. We suggest that this was partly due to quenching of fluorescence due to [H⁺], responsible for P to S (T) decay in control plants (Briantais et al. 1979) being replaced by state transition (state 2 to state 1) in dimethoate-treated plants (Kaňa et al. 2012).     

Prompt chlorophyll fluorescence as a tool for crop phenotyping: an example of barley landraces exposed to various abiotic stress factors

The study examined photosynthetic efficiency of two barley landraces (cvs. Arabi Abiad and Arabi Aswad) through a prompt fluorescence technique under influence of 14 different abiotic stress factors. The difference in the behavior of photosynthetic parameters under the same stress factor in–between cv. Arabi Abiad and cv. Arabi Aswad indicated different mechanisms of tolerance and strategies for the conversion of light energy into chemical energy for both the landraces. This study confirmed the suitability of some chlorophyll fluorescence parameters as reliable biomarkers for screening the plants at the level of photosynthetic apparatus.     

Steady-state chlorophyll fluorescence (Fs) measurements as a tool to follow variations of net CO2 assimilation and stomatal conductance during water-stress in C3 plants

Water stress experiments were performed with grapevines ( Vitis vinifera L.) and other C₃ plants in the field, in potted plants in the laboratory, and with detached leaves. It was found that, in all cases, the ratio of steady state chlorophyll fluorescence (Fs) normalized to dark-adapted intrinsic fluorescence (Fo) inversely correlated with non-photochemical quenching (NPQ). Also, at high irradiance, the ratio Fs/Fo was positively correlated with CO₂ assimilation in air, with electron transport rate calculated from fluorescence, and with stomatal conductance, but no clear correlation was observed with qP. The significance of these relationships is discussed. The ratio Fs/Fo, measured with a portable instrument (PAM-2000) or with a remote sensing FIPAM system, provides a good method for the early detection of water stress, and may become a useful guide to irrigation requirements.     

Kinetics of the oxygen evolution step in plants determined from flash-induced chlorophyll t a fluorescence

Photosystem II (PS II) of plants and cyanobacteria, which catalyzes the light-induced splitting of water and the release of oxygen, is the primary source of oxygen in the earth atmosphere. When activated by short light flashes, oxygen release in PS II occurs periodically with maxima after the third and the seventh flashes. Many other processes, including chlorophyll (Chl) t a fluorescence, are also modulated with period of four, reflecting their sensitivity to the activity of Photosystem II. A new approach has been developed for the analysis of the flash-induced fluorescence of Chl t a in plants, which is based on the use of the generalized Stern–Volmer equation for multiple quenchers. When applied to spinach thylakoids, this analysis reveals the presence of a new quencher of fluorescence whose amplitude is characterized by a periodicity of four with maxima after the third and the seventh flashes, in phase with oxygen release. The quencher appears with a delay of 0.5 ms followed by a rise time of 1.2–2 ms at pH 7, also in agreement with the expected time for oxygen evolution. It is concluded that the quencher is a product of the reaction leading to the oxygen evolution in PS II. The same quenching activity, maximal after the third flash, could be seen in dark adapted leaves, and provides the first fully time-resolved measurement of the kinetics of the oxygen evolution step in the leaf. Thus, the non-invasive probe of Chl t a fluorescence provides a new and sensitive method for measuring the kinetics of oxygen evolution with potential for use in plants and cyanobacteria t in vivo.     

Evaluation of chlorophyll fluorescence as a probe for drought stress in willow leaves

The effect of drought on the photosynthetic apparatus of leaves of Salix sp. was studied by measurements of the induction of chlorophyll fluorescence and the capacity for O₂ evolution. Using a multivariate analysis, a model was developed that could predict the degree of drought stress from the data of fluorescence kinetics. Even mild drought stress was detected with high precision; this was not always possible when the photosynthetic capacity was measured. The most clear discrimination between control and drought-stressed leaves was obtained if fluorescence induction was measured at high rather than normal CO₂ levels, and at low rather than high light levels. All information provided by fluorescence pertaining to drought was contained within the slow phase of the induction curve. It is suggested that rapid dehydration is different from drought at the mechanistic level as judged by the fluorescence characteristics.