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Plant leaves grow and change their orientation as well their emission of chlorophyll fluorescence in time. All these dynamic plant properties can be semi-automatically monitored by a 3D imaging system that generates plant models by the method of coded light illumination, fluorescence imaging and computer 3D reconstruction. Here, we describe the essentials of the method, as well as the system hardware. We show that the technique can reconstruct, with a high fidelity, the leaf size, the leaf angle and the plant height. The method fails with wilted plants when leaves overlap obscuring their true area. This effect, naturally, also interferes when the method is applied to measure plant growth under water stress. The method is, however, very potent in capturing the plant dynamics under mild stress and without stress. The 3D reconstruction is also highly effective in correcting geometrical factors that distort measurements of chlorophyll fluorescence emission of naturally positioned plant leaves.

Non-invasive, high-throughput screening methods are valuable tools in breeding for abiotic stress tolerance in plants. Optical signals such as chlorophyll fluorescence emission can be instrumental in developing new screening techniques. In order to examine the potential of chlorophyll fluorescence to reveal plant tolerance to low temperatures, we used a collection of nine Arabidopsis thaliana accessions and compared their fluorescence features with cold tolerance quantified by the well established electrolyte leakage method on detached leaves. We found that, during progressive cooling, the minimal chlorophyll fluorescence emission rose strongly and that this rise was highly dependent on the cold tolerance of the accessions. Maximum quantum yield of PSII photochemistry and steady state fluorescence normalized to minimal fluorescence were also highly correlated to the cold tolerance measured by the electrolyte leakage method. In order to further increase the capacity of the fluorescence detection to reveal the low temperature tolerance, we applied combinatorial imaging that employs plant classification based on multiple fluorescence features. We found that this method, by including the resolving power of several fluorescence features, can be well employed to detect cold tolerance already at mild sub-zero temperatures. Therefore, there is no need to freeze the screened plants to the largely damaging temperatures of around -15°C. This, together with the method's easy applicability, represents a major advantage of the fluorescence technique over the conventional electrolyte leakage method.

Rapid nondestructive screening of mutants is a common step in many research projects in plant biology. Here we report the development of a method that uses kinetic imaging of chlorophyll fluorescence to detect phenotypes that differ from wild-type plants. The method uses multiple fluorescence features simultaneously in order to catch different types of photosynthesis-related mutants with a single assay. The Mahalanobis distance was used to evaluate the degree of similarity in fluorescence features between the wild-type and test plants, and plants differing strongly from the wild-type were classified as mutants. The method was tested on a collection of photosynthesis-related mutants of Arabidopsis thaliana. The plants were evaluated from images in which the color of each pixel depended on the Mahalanobis distance of the fluorescence features. Two parameters of the color-coding procedure were used to adjust the trade-off between detection of true mutants and erratic classification of wild-type plants as mutants. We found that a large percentage of photosynthesis-related mutants can be detected with this method. Scripts for the free statistics software R are provided to facilitate the practical application of the method.

Automatic discrimination of plant species is required for precision farming and for advanced environmental protection. For this task, reflected sunlight has already been tested whereas fluorescence emission has been only scarcely considered. Here, we investigated the discriminative potential of chlorophyll fluorescence imaging in a case study using three closely related plant species of the family Lamiaceae. We compared discriminative potential of eight classifiers and four feature selection methods to identify the fluorescence parameters that can yield the highest contrast between the species. Three plant species: Ocimum basilicum, Origanum majorana and Origanum vulgare were grown separately as well as in pots where all three species were mixed. First, eight statistical classifiers were applied and tested in simulated species discrimination. The performance of the Quadratic Discriminant Classifier was found to be the most efficient. This classifier was further applied in combination with four different methods of feature selection. The Sequential Forward Floating Selection was found as the most efficient method for selecting the best performing subset of fluorescence images. The ability of the combinatorial statistical techniques for discriminating the species was also compared to the resolving power of conventional fluorescence parameters and found to be more efficient.

Plants are exposed to a temporally and spatially heterogeneous environment, and photosynthesis is well adapted to these fluctuations. Understanding of the complex, non-linear dynamics of photosynthesis in fluctuating light requires novel-modeling approaches that involve not only the primary light and dark biochemical reactions, but also networks of regulatory interactions. This requirement exceeds the capacity of the existing molecular models that are typically reduced to describe a partial process, dynamics of a specific complex or its particular dynamic feature. We propose a concept of comprehensive model that would represent an internally consistent, integral framework combining information on the reduced models that led to its construction. This review explores approaches and tools that exist in engineering, mathematics, and in other domains of biology that can be used to develop a comprehensive model of photosynthesis. Equally important, we investigated techniques by which one can rigorously reduce such a comprehensive model to models of low dimensionality, which preserve dynamic features of interest and, thus, contribute to a better understanding of photosynthesis under natural and thus fluctuating conditions. The web-based platform www.e-photosynthesis.org is introduced as an arena where these concepts and tools are being introduced and tested.

Pathogen infection leads to defence induction as well as to changes in carbohydrate metabolism of plants. Salicylic acid and oxylipins are involved in the induction of defence, but it is not known if these signalling molecules also mediate changes in carbohydrate metabolism. In this study, the effect of application of salicylic acid and the oxylipins 12-oxo-phytodienoic acid (OPDA) and jasmonic acid on photosynthesis was investigated by kinetic chlorophyll fluorescence imaging and compared with the effects of infection by virulent and avirulent strains of Pseudomonas syringae. Both pathogen strains and OPDA caused a similar change in fluorescence parameters of leaves of Arabidopsis thaliana. The response to OPDA appeared faster compared with that to the pathogens and persisted only for a short time. Infiltration with jasmonic acid or salicylic acid did not lead to a localized and distinct fluorescence response of the plant. To capture the faint early symptoms of the plant response, a novel algorithm was applied identifying the unique fluorescence signature-the set of images that, when combined, yield the highest contrast between control and infected leaf segments. Unlike conventional fluorescence parameters, this non-biased approach indeed detected the infection as early as 6 h after inoculation with bacteria. It was posssible to identify distinct fluorescence signatures characterizing the early and late phases of the infection. Fluorescence signatures of both infection phases were found in leaves infiltrated with OPDA.

Localized infection of a plant can be mapped by a sequence of images capturing chlorophyll fluorescence transients in actinic light. Choice of the actinic light protocol co-determines fluorescence contrast between infected leaf segment and surrounding healthy tissue. Frequently, biology cannot predict with which irradiance protocol, in which fluorescence image of the sequence, and in which segment of the image there will be the highest contrast between the healthy and infected tissue. Here, we introduce a new technique that can be applied to identify the combination of chlorophyll fluorescence images yielding the highest contrast. The sets of the most contrasting images vary throughout the progress of the infection. Such specific image sets, stress-revealing fluorescence signatures, can be found for the initial and late phases of the infection. Using these signatures, images can be divided into segments that show tissue in different infection phases. We demonstrate the capacity of the algorithm in an investigation of infection of the model plant Arabidopsis thaliana by the bacterium Pseudomonas syringae. We show that the highest contrast is found with transients elicited by fluctuating, harmonically modulated irradiance with long periods.

Photosynthetic organisms live in a dynamic environment where light typically fluctuates around a mean level that is slowly drifting during the solar day. We show that the far-from-equilibrium photosynthesis occurring in a rapidly fluctuating light differs vastly from the stationary-flux photosynthesis attained in a constant or slowly drifting light. Photosynthetic organisms in a static or slowly drifting light can be characterized by a steady-state quantum yield of chlorophyll fluorescence emission F' that is changing linearly with small and slow variations of the incident irradiance I+DeltaI(t): F'(I+DeltaI(t)) approximately Fmean '(dF)/(dI).DeltaI(t). In Synechocystis sp. PCC 6803, the linear approximation holds for an extended interval covering largely the static irradiance range experienced by the cyanobacteria in nature. The photosynthetic dynamism and, consequently, the dynamism of the chlorophyll fluorescence emission change dramatically when exposing the organism to a fluctuating irradiance. Harmonically-modulated irradiance I+DeltaI . sin(2pit/T), T approximately 1-25 s induces perpetual, far-from-equilibrium forced oscillations that are strongly non-linear, exhibiting significant hysteresis with multiple fluorescence levels corresponding to a single instantaneous level of the incident irradiance. We propose that, in nature, the far-from-equilibrium dynamic phenomena represent a significant correction to the steady-state photosynthetic activity that is typically investigated in laboratory. Analysis of the forced oscillations by the tools of systems biology suggests that the dynamism of photosynthesis observed in fluctuating light can be explained by a delayed action of regulatory agents.

Photosynthetic organisms exposed to a dynamic light environment exhibit complex transients of photosynthetic activities that are strongly dependent on the temporal pattern of the incident irradiance. In a harmonically modulated light of intensity I approximately const.+sin(omegat), chlorophyll fluorescence response consists of a steady-state component, a component modulated with the angular frequency of the irradiance omega and several upper harmonic components (2omega, 3omega and higher). Our earlier reverse engineering analysis suggests that the non-linear response can be caused by a negative feedback regulation of photosynthesis. Here, we present experimental evidence that the negative feedback regulation of the energetic coupling between phycobilisome and Photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC6803 indeed results in the appearance of upper harmonic modes in the chlorophyll fluorescence emission. Dynamic changes in the coupling of the phycobilisome to PSII are not accompanied by corresponding antiparallel changes in the Photosystem I (PSI) excitation, suggesting a regulation limited to PSII. Strong upper harmonic modes were also found in the kinetics of the non-photochemical quenching (NPQ) of chlorophyll fluorescence, of the P700 redox state and of the CO(2) assimilation in tobacco (Nicotiana tabaccum) exposed to harmonically modulated light. They are ascribed to negative feedback regulation of the reactions of the Calvin-Benson cycle limiting the photosynthetic electron transport. We propose that the observed non-linear response of photosynthesis may also be relevant in a natural light environment that is modulated, e.g., by ocean waves, moving canopy or by varying cloud cover. Under controlled laboratory conditions, the non-linear photosynthetic response provides a new insight into dynamics of the regulatory processes.

Chlorophyll fluorescence kinetic microscopy was used to analyze photosynthetic oscillations in individual cells of leaves and in isolated leaf cell protoplasts. Four Brassicaceae species were used: Arabidopsis halleri (L.) O'Kane & Al-Shehbaz, Thlaspi fendleri (Nels.) Hitchc, Thlaspi caerulescens J.&C. Presl and Thlaspi ochroleucum Boiss et Helder. With the latter two, the measurements were extended also to isolated protoplasts. The oscillations were induced under the microscope by exposing dark-adapted samples to actinic irradiance. Detailed analysis of the induced transients revealed that they consist of several processes oscillating with different frequencies and not only one component as reported earlier. Furthermore, it was found that most of these processes are controlled inside each individual cell. This was shown by differences in oscillations in neighboring cells and protoplasts that share a uniform intercellular environment. The frequency of the dominant oscillation frequency depended neither on irradiance nor on CO2 concentration and is, therefore, not controlled by the photosynthetic rate. Characteristic differences in the frequency spectrum and damping of oscillations have been found among the plant species examined.