From leaf to multiscale models of photosynthesis: applications and challenges for crop improvement.

To keep up with the growth of human population and to circumvent deleterious effects of global climate change, it is essential to enhance crop yield to achieve higher production. Here we review mathematical models of oxygenic photosynthesis that are extensively used, and discuss in depth a subset that accounts for diverse approaches providing solutions to our objective. These include models (1) to study different ways to enhance photosynthesis, such as fine-tuning antenna size, photoprotection and electron transport; (2) to bioengineer carbon metabolism; and (3) to evaluate the interactions between the process of photosynthesis and the seasonal crop dynamics, or those that have included statistical whole-genome prediction methods to quantify the impact of photosynthesis traits on the improvement of crop yield. We conclude by emphasizing that the results obtained in these studies clearly demonstrate that mathematical modelling is a key tool to examine different approaches to improve photosynthesis for better productivity, while effective multiscale crop models, especially those that also include remote sensing data, are indispensable to verify different strategies to obtain maximized crop yields.

Dynamics and interplay of photosynthetic regulatory processes depend on the amplitudes of oscillating light.

Plants have evolved multiple regulatory mechanisms to cope with natural light fluctuations. The interplay between these mechanisms leads presumably to the resilience of plants in diverse light patterns. We investigated the energy-dependent nonphotochemical quenching (qE) and cyclic electron transports (CET) in light that oscillated with a 60-s period with three different amplitudes. The photosystem I (PSI) and photosystem II (PSII) function-related quantum yields and redox changes of plastocyanin and ferredoxin were measured in Arabidopsis thaliana wild types and mutants with partial defects in qE or CET. The decrease in quantum yield of qE due to the lack of either PsbS- or violaxanthin de-epoxidase was compensated by an increase in the quantum yield of the constitutive nonphotochemical quenching. The mutant lacking NAD(P)H dehydrogenase (NDH)-like-dependent CET had a transient significant PSI acceptor side limitation during the light rising phase under high amplitude of light oscillations. The mutant lacking PGR5/PGRL1-CET restricted electron flows and failed to induce effective photosynthesis control, regardless of oscillation amplitudes. This suggests that PGR5/PGRL1-CET is important for the regulation of PSI function in various amplitudes of light oscillation, while NDH-like-CET acts' as a safety valve under fluctuating light with high amplitude. The results also bespeak interplays among multiple photosynthetic regulatory mechanisms.

Fluorescence to measure light intensity.

Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.

Plants cope with fluctuating light by frequency-dependent nonphotochemical quenching and cyclic electron transport.

In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light. Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport. The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics. Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.

Effect of ion fluxes on regulating the light-induced transthylakoid electric potential difference.

The light-induced transthylakoid membrane potential (ΔΨ) can not only drive the ATP synthesis through the ATP-synthase in chloroplasts but serve as an essential modifier in the acclimation of photosynthesis to fluctuating light conditions. It has been manifested that during photosynthesis, the light-induced ΔΨ is responsive to multiple factors among which the ion channels/transporters (e.g., V-K+, VCCN1, and KEA3) are key to adjust the ion distribution on the two sides of the thylakoid membrane and hence shape the kinetics of ΔΨ. However, an in-depth mechanistic understanding of ion fluxes on adjusting the transthylakoid electric potentials is still unclear. This lack of a mechanistic understanding is due to the experimental difficulty of closely observing ion fluxes in vivo and also hacking the evolution of parameters in a highly intertwined photosynthetic network. In this work, a computer model was applied to investigate the roles of ion fluxes on adjusting transthylakoid electric potentials upon a temporal cycle of a period of high illumination followed by a dark-adapted phase. The computing data revealed that, firstly, upon illumination, the dissipation of the steady-ΔΨ by ∼10 mV is contributed from the V-K+-driven K+ flux whilst ∼8 mV of the steady-ΔΨ is dissipated by the VCCN1-pumped Cl- flux, but there were no appreciable KEA3-evoked variations on ΔΨ; secondly, on transition from high light to darkness, V-K+ and KEA3 are serving as major contributors whereas VCCN1 taking a counterbalancing part in shaping a standard trace of ECS (electrochromic shift), which commonly shows a sharp fall to a minimum before returning to the baseline in darkness. Besides, the functional consequences on components of ΔΨ adjusted by every particular ion channel/transporter were also explored. By employing the model, we bring evidence that particular thylakoid-harbored proteins, namely V-K+, VCCN1, and KEA3, function by distinct mechanisms in the dynamic adjustment of electric potential, which might be mainly importnat under fluctuating light conditions.

Analyzing the effect of ion binding to the membrane-surface on regulating the light-induced transthylakoid electric potential (ΔΨm).

The transthylakoid membrane potential (ΔΨm) is essential because it can drive the ATP synthesis through the CF0-CF1 type of ATP-synthase in chloroplasts as an energetic equivalent similar to ΔpH. In addition, a high fraction of proton motive force (PMF) stored as the ΔΨm component is physiologically important in the acclimation of photosynthesis to environmental stresses. It has been shown that ΔΨm is the sum of the Donnan potential difference (ΔΨdn) and the diffusion potential difference (ΔΨd). Specifically, ΔΨdn, ΔΨd, and ΔΨm are strongly associated with the ionic activities near the membrane surface, particularly, the extent of ion binding to the charged/neutral sites adjacent to the membrane surface. However, an in-depth analysis of the effect of altered cationic binding to the membrane surface on adjusting the transthylakoid electric potentials (ΔΨdn, ΔΨd, and ΔΨm) is still missing. This lack of a mechanistic understanding is due to the experimental difficulty of closely observing cations binding to the membrane surface in vivo. In this work, a computer model was proposed to investigate the transthylakoid electric phenomena in the chloroplast focusing on the interaction between cations and the negative charges close to the membrane surface. By employing the model, we simulated the membrane potential and consequently, the measured ECS traces, proxing the ΔΨm, were well described by the computing results on continuous illumination followed by a dark-adapted period. Moreover, the computing data clarified the components of transthylakoid membrane potential, unraveled the functional consequences of altered cationic attachment to the membrane surface on adjusting the transthylakoid electric potential, and further revealed the key role played by Donnan potential in regulating the energization of the thylakoid membrane. The current model for calculating electric potentials can function as a preliminary network for the further development into a more detailed theoretical model by which multiple important variables involved in photosynthesis can be explored.

Insights on the regulation of photosynthesis in pea leaves exposed to oscillating light.

Plants growing in nature often experience fluctuating irradiance. However, in the laboratory, the dynamics of photosynthesis are usually explored by instantaneously exposing dark-adapted plants to constant light and examining the dark-to-light transition, which is a poor approximation of natural phenomena. With the aim creating a better approximation, we exposed leaves of pea (Pisum sativum) to oscillating light and measured changes in the functioning of PSI and PSII, and of the proton motive force at the thylakoid membrane. We found that the dynamics depended on the oscillation period, revealing information about the underlying regulatory networks. As demonstrated for a selected oscillation period of 60 s, the regulation tries to keep the reaction centers of PSI and PSII open. We present an evaluation of the data obtained, and discuss the involvement of particular processes in the regulation of photosynthesis. The forced oscillations provided an information-rich fingerprint of complex regulatory networks. We expect future progress in understanding these networks from experiments involving chemical interventions and plant mutants, and by using mathematical modeling and systems identification and control tools.

Photosynthesis dynamics and regulation sensed in the frequency domain.

Foundations of photosynthesis research have been established mainly by studying the response of plants to changing light, typically to sudden exposure to a constant light intensity after dark acclimation or light flashes. This approach remains valid and powerful, but can be limited by requiring dark acclimation before time-domain measurements and often assumes that rate constants determining the photosynthetic response do not change between dark and light acclimation. We show that these limits can be overcome by measuring plant responses to sinusoidally modulated light of varying frequency. By its nature, such frequency-domain characterization is performed in light-acclimated plants with no need for prior dark acclimation. Amplitudes, phase shifts, and upper harmonic modulation extracted from the data for a wide range of frequencies can target different kinetic domains and regulatory feedbacks. The occurrence of upper harmonic modulation reflects nonlinear phenomena, including photosynthetic regulation. To support these claims, we measured chlorophyll fluorescence emission of the green alga Chlorella sorokiniana in light that was sinusoidally modulated in the frequency range 1000-0.001 Hz. Based on these experimental data and numerical as well as analytical mathematical models, we propose that frequency-domain measurements can become a versatile tool in plant sensing.

Towards spruce-type photosystem II: consequences of the loss of light-harvesting proteins LHCB3 and LHCB6 in Arabidopsis.

The largest stable photosystem II (PSII) supercomplex in land plants (C2S2M2) consists of a core complex dimer (C2), two strongly (S2) and two moderately (M2) bound light-harvesting protein (LHCB) trimers attached to C2 via monomeric antenna proteins LHCB4-6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C2S2M2 supercomplex. Here, we performed structure-function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the "spruce-type" PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The "spruce-type" PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C2S2 semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C2S2 arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.

Reconstruction of the absorption spectrum of Synechocystis sp. PCC 6803 optical mutants from the in vivo signature of individual pigments.

In this work, we reconstructed the absorption spectrum of different Synechocystis sp. PCC 6803 optical strains by summing the computed signature of all pigments present in this organism. To do so, modifications to in vitro pigment spectra were first required: namely wavelength shift, curve smoothing, and the package effect calculation derived from high pigment densities were applied. As a result, we outlined a plausible shape for the in vivo absorption spectrum of each chromophore. These are flatter and slightly broader in physiological conditions yet the mean weight-specific absorption coefficient remains identical to the in vitro conditions. Moreover, we give an estimate of all pigment concentrations without applying spectrophotometric correlations, which are often prone to error. The computed cell spectrum reproduces in an accurate manner the experimental spectrum for all the studied wavelengths in the wild-type, Olive, and PAL strain. The gathered pigment concentrations are in agreement with reported values in literature. Moreover, different illumination set-ups were evaluated to calculate the mean absorption cross-section of each chromophore. Finally, a qualitative estimate of light-limited cellular growth at each wavelength is given. This investigation describes a novel way to approach the cell absorption spectrum and shows all its inherent potential for photosynthesis research.

Photosynthetic performance of Antarctic lichen Dermatocarpon polyphyllizum when affected by desiccation and low temperatures.

Lichens are symbiotic organisms that are well adapted to desiccation/rehydration cycles. Over the last decades, the physiological background of their photosynthetic response-specifically activation of the protective mechanism during desiccation-has been studied at the level of photosystem II of the lichen photobiont by means of several biophysical methods. In our study, the effects of desiccation and low temperatures on chlorophyll fluorescence and spectral reflectance parameters were investigated in Antarctic chlorolichen Dermatocarpon polyphyllizum. Lichen thalli were collected from James Ross Island, Antarctica, and following transfer to a laboratory, samples were fully hydrated and exposed to desiccation at temperatures of 18, 10, and 4 °C. During the desiccation process, the relative water content (RWC) was measured gravimetrically and photosynthetic parameters related to the fast transient of chlorophyll fluorescence (OJIP) were measured repeatedly. Similarly, the change in spectral reflectance parameters (e.g., NDVI, PRI, G, NPCI) was monitored during thallus dehydration. The dehydration-response curves showed a decrease in a majority of the OJIP-derived parameters (e.g., maximum quantum yield of photosystem II photochemistry: FV/FM, and performance index: PI in D. polyphyllizum, which were more apparent at RWCs below 20%. The activation of protective mechanisms in severely dehydrated thalli was documented by increased thermal dissipation (DI0/RC) and its quantum yield (Phi_D0). Low temperature accelerated these processes. An analysis of the OJIP shape reveals the presence of K-bands (300 µs), and L-bands (80 µs), which can be attributed to dehydration-induced stress. Spectral reflectance indices decreased in a majority of cases with an RWC decrease and were positively related to the OJIP-derived parameters: FV/FM (capacity of photosynthetic processes in PSII), Phi_E0 (effectiveness of electron transport), and PI_tot (total performance index), which was more apparent in NDVI. A negative relation was found for NPCI. These indices could be used in follow-up ecophysiological photosynthetic studies of lichens that are undergoing rehydration/dehydration cycles.

Photosynthesis: basics, history and modelling.

BACKGROUND: With limited agricultural land and increasing human population, it is essential to enhance overall photosynthesis and thus productivity. Oxygenic photosynthesis begins with light absorption, followed by excitation energy transfer to the reaction centres, primary photochemistry, electron and proton transport, NADPH and ATP synthesis, and then CO2 fixation (Calvin-Benson cycle, as well as Hatch-Slack cycle). Here we cover some of the discoveries related to this process, such as the existence of two light reactions and two photosystems connected by an electron transport 'chain' (the Z-scheme), chemiosmotic hypothesis for ATP synthesis, water oxidation clock for oxygen evolution, steps for carbon fixation, and finally the diverse mechanisms of regulatory processes, such as 'state transitions' and 'non-photochemical quenching' of the excited state of chlorophyll a. SCOPE: In this review, we emphasize that mathematical modelling is a highly valuable tool in understanding and making predictions regarding photosynthesis. Different mathematical models have been used to examine current theories on diverse photosynthetic processes; these have been validated through simulation(s) of available experimental data, such as chlorophyll a fluorescence induction, measured with fluorometers using continuous (or modulated) exciting light, and absorbance changes at 820 nm (ΔA820) related to redox changes in P700, the reaction centre of photosystem I. CONCLUSIONS: We highlight here the important role of modelling in deciphering and untangling complex photosynthesis processes taking place simultaneously, as well as in predicting possible ways to obtain higher biomass and productivity in plants, algae and cyanobacteria.

Exogenous application of cytokinin during dark senescence eliminates the acceleration of photosystem II impairment caused by chlorophyll b deficiency in barley.

Recent studies have shown that chlorophyll (Chl) b has an important role in the regulation of leaf senescence. However, there is only limited information about senescence of plants lacking Chl b and senescence-induced decrease in photosystem II (PSII) and photosystem I (PSI) function has not even been investigated in such plants. We have studied senescence-induced changes in photosynthetic pigment content and PSII and PSI activities in detached leaves of Chl b-deficient barley mutant, chlorina f2f2 (clo). After 4 days in the dark, the senescence-induced decrease in PSI activity was smaller in clo compared to WT leaves. On the contrary, the senescence-induced impairment in PSII function (estimated from Chl fluorescence parameters) was much more pronounced in clo leaves, even though the relative decrease in Chl content was similar to wild type (WT) leaves (Hordeum vulgare L., cv. Bonus). The stronger impairment of PSII function seems to be related to more pronounced damage of reaction centers of PSII. Interestingly, exogenously applied plant hormone cytokinin 6-benzylaminopurine (BA) was able to maintain PSII function in the dark senescing clo leaves to a similar extent as in WT. Thus, considering the fact that without BA the senescence-induced decrease in PSII photochemistry in clo was more pronounced than in WT, the relative protective effect of BA was higher in Chl b-deficient mutant than in WT.

Heart failure disease management program, its contribution to established pharmacotherapy and long-term prognosis in real clinical practice - retrospective data analysis.

BACKGROUND AND AIMS: The prognosis of patients with heart failure (HF) is still generally unfavorable. HF with reduced ejection fraction (HFrEF) patients reach target medication doses in very low percentages in daily clinical practice. HF disease management programs (DMP), including nurse and telemedicine support that facilitate achieving target medication doses, may improve the unfavorable prognosis. METHODS: We retrospectively analyzed the data of 738 patients with HFrEF who were followed in a single HF center during the years 1975-2011, for 6.4 (median) years. DMP, nurse and telemedicine support is established at this center. RESULTS: The group achieved left ventricle (LV) recovery after the HF treatment. The median LV ejection fraction improved from 25.0% at baseline to 50.0% at the time of the latest data collection. The proportion of NYHA II, III and IV classes decreased from 27.6%, 30.2% and 29.7% to 26.6%, 7.2% and 0.1%, respectively while the proportion of NYHA class I increased from 12.5% to 66.1%. Median NT-proBNP decreased from 975.0 to 324.0 pg/mL. The survival of the patient group was favorable; 79.7% survived 18.1 years after diagnosis of HF. A high percentage of the patients received recommended target or higher than target doses of angiotensin-converting enzyme inhibitors (82.0%) and beta-blockers (78.1%). CONCLUSION: The established pharmacotherapy resulted from an effective DMP and this contributed to the favorable prognosis.

On the source of non-linear light absorbance in photosynthetic samples.

This study presents a mathematical model, which expresses the absorbance of a photosynthetic sample as a non-linear polynomial of selected reference absorbance. The non-linearity is explained by inhomogeneities of a product of pigment concentration and light path length in the sample. The quadratic term of the polynomial reflects the extent of inhomogeneities, and the cubic term is related to deviation of the product distribution from a symmetric one. The model was tested by measurements of suspension of unstacked tobacco thylakoid membranes of different chlorophyll concentrations in cuvettes of different thicknesses. The absorbance was calculated from the diffuse transmittance and reflectance of sample, illuminated by perpendicular collimated light. The evaluated quantity was a sensitivity defined as the relative difference between the sample absorbance and the reference absorbance to the reference absorbance. The non-linearity of sample absorbance was demonstrated by a characteristic deviation of the sensitivity spectrum from a constant value. The absorbance non-linearity decreased on an increase of the product of pigment concentration and cuvette thickness. The model suggests that the sieve and detour effects influence the absorbance in a similar way. The model may be of interest in modeling of leaf or canopy optics including light absorption and scattering.

Modeling the light-induced electric potential difference (ΔΨ), the pH difference (ΔpH) and the proton motive force across the thylakoid membrane in C3 leaves.

A model was constructed which includes electron transport (linear and cyclic and Mehler type reaction) coupled to proton translocation, counter ion movement, ATP synthesis, and Calvin-Benson cycle. The focus is on modeling of the light-induced total electric potential difference (ΔΨ) which in this model originates from the bulk phase electric potential difference (ΔΨb), the localized electric potential difference (ΔΨc), as well as the surface electric potential difference (ΔΨs). The measured dual wavelength transmittance signal (ΔA515-560nm, electrochromic shift) was used as a proxy for experimental ΔΨ. The predictions for theoretical ΔΨ vary with assumed contribution of ΔΨs, which might imply that the measured ΔA515-560nm trace on a long time scale reflects the interplay of the ΔΨ components. Simulations also show that partitioning of proton motive force (pmf) to ΔΨb and ΔpH components is sensitive to the stoichiometric ratio of H+/ATP, energy barrier for ATP synthesis, ionic strength, buffer capacity and light intensity. Our model shows that high buffer capacity promotes the establishment of ΔΨb, while the formation of pHi minimum is not 'dissipated' but 'postponed' until it reaches the same level as that for low buffer capacity. Under physiologically optimal conditions, the output of the model shows that at steady state in light, the ΔpH component is the main contributor to pmf to drive ATP synthesis while a low ΔΨb persists energizing the membrane. Our model predicts 11mV as the resting electric potential difference across the thylakoid membrane in dark. We suggest that the model presented in this work can be integrated as a module into a more comprehensive model of oxygenic photosynthesis.

Modeling the light-induced electric potential difference ΔΨ across the thylakoid membrane based on the transition state rate theory.

In photosynthesis, electron transport-coupled proton movement initiates the formation of the light-induced electric potential difference, ΔΨ, across the thylakoid membrane (TM). Ions are transported across the TM to counterbalance the charge of protons accumulated in the lumen. The objective of this work is to construct range of mathematical models for simulation of ΔΨ, using the transition state rate theory (TSRT) for description of movement of ions through the channels. The TSRT considers either single-ion (TSRT-SI) or multi-ion occupancy (TSRT-MI) in the channels. Movement of ions through the channel pore is described by means of energy barriers and binding sites; ions move in and out of vacant sites with rate constants that depend on the barrier heights and well depths, as well as on the interionic repulsion in TSRT-MI model. Three energy motifs are used to describe the TSRT-SI model: two-barrier one-site (2B1S), three-barrier two-site (3B2S), and four-barrier three-site (4B3S). The 3B2S energy motif is used for the TSRT-MI model. The accumulation of cations due to the TM surface negative fixed charges is also taken into account. A model employing the electro-diffusion theory instead of the TSRT is constructed for comparison. The dual wavelength transmittance signal (ΔA515-560nm) measuring the electrochromic shift (ECS) provides a proxy for experimental light-induced ΔΨ. The simulated ΔΨ traces qualitatively agree with the measured ECS traces. The models can simulate different channel conducting regimes and assess their impact on ΔΨ. The ionic flux coupling in the TSRT-MI model suggests that an increase in the internal or external K+ concentration may block the outward or the inward Mg2+ current, respectively.

Automated phenotyping of plant shoots using imaging methods for analysis of plant stress responses - a review.

Current methods of in-house plant phenotyping are providing a powerful new tool for plant biology studies. The self-constructed and commercial platforms established in the last few years, employ non-destructive methods and measurements on a large and high-throughput scale. The platforms offer to certain extent, automated measurements, using either simple single sensor analysis, or advanced integrative simultaneous analysis by multiple sensors. However, due to the complexity of the approaches used, it is not always clear what such forms of plant phenotyping can offer the potential end-user, i.e. plant biologist. This review focuses on imaging methods used in the phenotyping of plant shoots including a brief survey of the sensors used. To open up this topic to a broader audience, we provide here a simple introduction to the principles of automated non-destructive analysis, namely RGB, chlorophyll fluorescence, thermal and hyperspectral imaging. We further on present an overview on how and to which extent, the automated integrative in-house phenotyping platforms have been used recently to study the responses of plants to various changing environments.

Automated integrative high-throughput phenotyping of plant shoots: a case study of the cold-tolerance of pea (Pisum sativum L.).

BACKGROUND: Recently emerging approaches to high-throughput plant phenotyping have discovered their importance as tools in unravelling the complex questions of plant growth, development and response to the environment, both in basic and applied science. High-throughput methods have been also used to study plant responses to various types of biotic and abiotic stresses (drought, heat, salinity, nutrient-starving, UV light) but only rarely to cold tolerance. RESULTS: We present here an experimental procedure of integrative high-throughput in-house phenotyping of plant shoots employing automated simultaneous analyses of shoot biomass and photosystem II efficiency to study the cold tolerance of pea (Pisum sativum L.). For this purpose, we developed new software for automatic RGB image analysis, evaluated various parameters of chlorophyll fluorescence obtained from kinetic chlorophyll fluorescence imaging, and performed an experiment in which the growth and photosynthetic activity of two different pea cultivars were followed during cold acclimation. The data obtained from the automated RGB imaging were validated through correlation of pixel based shoot area with measurement of the shoot fresh weight. Further, data obtained from automated chlorophyll fluorescence imaging analysis were compared with chlorophyll fluorescence parameters measured by a non-imaging chlorophyll fluorometer. In both cases, high correlation was obtained, confirming the reliability of the procedure described. CONCLUSIONS: This study of the response of two pea cultivars to cold stress confirmed that our procedure may have important application, not only for selection of cold-sensitive/tolerant varieties of pea, but also for studies of plant cold-response strategies in general. The approach, provides a very broad tool for the morphological and physiological selection of parameters which correspond to shoot growth and the efficiency of photosystem II, and is thus applicable in studies of various plant species and crops.