Polymer-Modified Single-Walled Carbon Nanotubes Affect Photosystem II Photochemistry, Intersystem Electron Transport Carriers and Photosystem I End Acceptors in Pea Plants.

Single-walled carbon nanotubes (SWCNT) have recently been attracting the attention of plant biologists as a prospective tool for modulation of photosynthesis in higher plants. However, the exact mode of action of SWCNT on the photosynthetic electron transport chain remains unknown. In this work, we examined the effect of foliar application of polymer-grafted SWCNT on the donor side of photosystem II, the intersystem electron transfer chain and the acceptor side of photosystem I. Analysis of the induction curves of chlorophyll fluorescence via JIP test and construction of differential curves revealed that SWCNT concentrations up to 100 mg/L did not affect the photosynthetic electron transport chain. SWCNT concentration of 300 mg/L had no effect on the photosystem II donor side but provoked inactivation of photosystem II reaction centres and slowed down the reduction of the plastoquinone pool and the photosystem I end acceptors. Changes in the modulated reflection at 820 nm, too, indicated slower re-reduction of photosystem I reaction centres in SWCNT-treated leaves. We conclude that SWCNT are likely to be able to divert electrons from the photosynthetic electron transport chain at the level of photosystem I end acceptors and plastoquinone pool in vivo. Further research is needed to unequivocally prove if the observed effects are due to specific interaction between SWCNT and the photosynthetic apparatus.

Exploration of Chlorophyll a Fluorescence and Plant Gas Exchange Parameters as Indicators of Drought Tolerance in Perennial Ryegrass.

Perennial ryegrass (Lolium perenne L.) belongs to the common cultivated grass species in Central and Western Europe. Despite being considered to be susceptible to drought, it is frequently used for forming the turf in urban green areas. In such areas, the water deficit in soil is recognized as one of the most important environmental factors, which can limit plant growth. The basic aim of this work was to explore the mechanisms standing behind the changes in the photosynthetic apparatus performance of two perennial ryegrass turf varieties grown under drought stress using comprehensive in vivo chlorophyll fluorescence signal analyses and plant gas exchange measurements. Drought was applied after eight weeks of sowing by controlling the humidity of the roots ground medium at the levels of 30, 50, and 70% of the field water capacity. Measurements were carried out at four times: 0, 120, and 240 h after drought application and after recovery (refilling water to 70%). We found that the difference between the two tested varieties' response resulted from a particular re-reduction of P700+ (reaction certer of PSI) that was caused by slower electron donation from P680. The difference in the rate of electron flow from Photosystem II (PSII) to PSI was also detected. The application of the combined tools (plants' photosynthetic efficiency analysis and plant gas exchange measurements) allowed exploring and explaining the specific variety response to drought stress.

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.

Effects of Different Metals on Photosynthesis: Cadmium and Zinc Affect Chlorophyll Fluorescence in Durum Wheat.

A comparative study of the effects of exposure to high Cd2+ (50 µM) and excess Zn2+ (600 µM) on photosynthetic performance of hydroponically-grown durum wheat seedlings was performed. At day 8, Cd and Zn were added to the nutrient solution. After 7-days exposure, the chosen concentrations of both metals resulted in similar relative growth rate (RGR) inhibitions of about 50% and comparable retardations of the CO2 assimilation rates (about 30%) in the second developed leaf of wheat seedlings. Analysis of chlorophyll a fluorescence indicated that both metals disturbed photosynthetic electron transport processes which led to a 4- to 5-fold suppression of the efficiency of energy transformation in Photosystem II. Non-specific toxic effects of Cd and Zn, which prevailed, were an inactivation of part of Photosystem II reaction centres and their transformation into excitation quenching forms as well as disturbed electron transport in the oxygen-evolving complex. The specificity of the Cd and Zn modes of action was mainly expressed in the intensity of the toxicity effects: despite the similar inhibitions of the CO2 assimilation rates, the wheat photochemistry showed much more sensitivity to Cd than to Zn exposure.

Frequently asked questions about chlorophyll fluorescence, the sequel.

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F V /F M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.

Frequently asked questions about in vivo chlorophyll fluorescence: practical issues.

The aim of this educational review is to provide practical information on the hardware, methodology, and the hands on application of chlorophyll (Chl) a fluorescence technology. We present the paper in a question and answer format like frequently asked questions. Although nearly all information on the application of Chl a fluorescence can be found in the literature, it is not always easily accessible. This paper is primarily aimed at scientists who have some experience with the application of Chl a fluorescence but are still in the process of discovering what it all means and how it can be used. Topics discussed are (among other things) the kind of information that can be obtained using different fluorescence techniques, the interpretation of Chl a fluorescence signals, specific applications of these techniques, and practical advice on different subjects, such as on the length of dark adaptation before measurement of the Chl a fluorescence transient. The paper also provides the physiological background for some of the applied procedures. It also serves as a source of reference for experienced scientists.

Drought-induced modifications of photosynthetic electron transport in intact leaves: analysis and use of neural networks as a tool for a rapid non-invasive estimation.

Water deficit is one of the most important environmental factors limiting sustainable crop yields and it requires a reliable tool for fast and precise quantification. In this work we use simultaneously recorded signals of photoinduced prompt fluorescence (PF) and delayed fluorescence (DF) as well as modulated reflection (MR) of light at 820nm for analysis of the changes in the photosynthetic activity in detached bean leaves during drying. Depending on the severity of the water deficit we identify different changes in the primary photosynthetic processes. When the relative water content (RWC) is decreased to 60% there is a parallel decrease in the ratio between the rate of excitation trapping in the Photosystem (PS) II reaction center and the rate of reoxidation of reduced PSII acceptors. A further decrease of RWC to 20% suppresses the electron transfer from the reduced plastoquinone pool to the PSI reaction center. At RWC below values 15%, the reoxidation of the photoreduced primary quinone acceptor of PSII, Q(A)(-), is inhibited and at less than 5%, the primary photochemical reactions in PSI and II are inactivated. Using the collected sets of PF, DF and MR signals, we construct and train an artificial neural network, capable of recognizing the RWC in a series of "unknown" samples with a correlation between calculated and gravimetrically determined RWC values of about R(2)≈0.98. Our results demonstrate that this is a reliable method for determination of RWC in detached leaves and after further development it could be used for quantifying of drought stress of crop plants in situ. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Experimental in vivo measurements of light emission in plants: a perspective dedicated to David Walker.

This review is dedicated to David Walker (1928-2012), a pioneer in the field of photosynthesis and chlorophyll fluorescence. We begin this review by presenting the history of light emission studies, from the ancient times. Light emission from plants is of several kinds: prompt fluorescence (PF), delayed fluorescence (DF), thermoluminescence, and phosphorescence. In this article, we focus on PF and DF. Chlorophyll a fluorescence measurements have been used for more than 80 years to study photosynthesis, particularly photosystem II (PSII) since 1961. This technique has become a regular trusted probe in agricultural and biological research. Many measured and calculated parameters are good biomarkers or indicators of plant tolerance to different abiotic and biotic stressors. This would never have been possible without the rapid development of new fluorometers. To date, most of these instruments are based mainly on two different operational principles for measuring variable chlorophyll a fluorescence: (1) a PF signal produced following a pulse-amplitude-modulated excitation and (2) a PF signal emitted during a strong continuous actinic excitation. In addition to fluorometers, other instruments have been developed to measure additional signals, such as DF, originating from PSII, and light-induced absorbance changes due to the photooxidation of P700, from PSI, measured as the absorption decrease (photobleaching) at about 705 nm, or increase at 820 nm. In this review, the technical and theoretical basis of newly developed instruments, allowing for simultaneous measurement of the PF and the DF as well as other parameters is discussed. Special emphasis has been given to a description of comparative measurements on PF and DF. However, DF has been discussed in greater details, since it is much less used and less known than PF, but has a great potential to provide useful qualitative new information on the back reactions of PSII electron transfer. A review concerning the history of fluorometers is also presented.

Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis.

A new instrument (M-PEA), which measures simultaneously kinetics of prompt fluorescence (PF), delayed fluorescence (DF) and modulated light reflection at 820nm (MR), was used to screen dark-adapted leaves of the resurrection plant Haberlea rhodopensis during their progressive drying, down to 1% relative water content (RWC), and after their re-watering. This is the first investigation using M-PEA, which employs alternations of actinic light (627-nm peak, 5000 micromol photons m(-2) s(-1)) and dark intervals, where PF-MR and DF kinetics are respectively recorded, with the added advantages: (a) all kinetics are recorded with high time resolution (starting from 0.01 ms), (b) the dark intervals' duration can be as short as 0.1 ms, (c) actinic illumination can be interrupted at different times during the PF transient (recorded up to 300 s), with the earliest interruption at 0.3 ms. Analysis of the simultaneous measurements at different water-content-states of H. rhodopensis leaves allowed the comparison and correlation of complementary information on the structure/function of the photosynthetic machinery, which is not destroyed but only inactivated (reversibly) at different degrees; the comparison and correlation helped also to test current interpretations of each signal and advance their understanding. Our results suggest that the desiccation tolerance of the photosynthetic machinery in H. rhodopensis is mainly based on mechanism(s) that lead to inactivation of photosystem II reaction centres (transformation to heat sinks), triggered already by a small RWC decrease.

Functional Characterization of the Photosynthetic Machinery in Smicronix Galls on the Parasitic Plant Cuscuta campestris by JIP-Test.

Members of the genus Cuscuta are generally considered to be non-photosynthetic, stem-holoparasitic flowering plants. Under certain circumstances, at least some members of the genus are capable of limited photosynthesis. The galls of the Smicronyx weevils formed on Cuscuta campestris are particularly rich in chlorophylls compared to the stem of the parasitic plant. In the present study, we aimed to characterize the photosynthetic activity in the inner and outer gall cortices in comparison to the non-photosynthetic stems and a reference plant (Arabidopsis thaliana). The recorded prompt chlorophyll fluorescence transients were analyzed using JIP test. Detailed analysis of the chlorophyll fluorescence confirmed the presence of actively functioning photosynthetic machinery, especially in the inner cortex of the galls. This photosynthesis, induced by the insect larvae, did not reach the levels of the photosynthetic activity in Arabidopsis thaliana plants. Thylakoid protein complexes were identified by separation with two-dimensional Blue Native/SDS PAGE. It appeared that some of the complexes presented in A. thaliana are missing in C. campestris. We hypothesize that the insect-triggered transition from non-photosynthetic to photosynthetic tissue in the gall is driven by the increased requirements for nutrients related to the larval nutrition.

Extraction of Proteins and Other Intracellular Bioactive Compounds From Baker's Yeasts by Pulsed Electric Field Treatment.

Yeasts are rich source of proteins, antioxidants, vitamins, and other bioactive compounds. The main drawback in their utilization as valuable ingredients in functional foods and dietary supplements production is the thick, indigestible cell wall, as well as the high nucleic acid content. In this study, we evaluated the feasibility of pulsed electric field (PEF) treatment as an alternative method for extraction of proteins and other bioactive intracellular compounds from yeasts. Baker's yeast water suspensions with different concentration (12.5-85 g dry cell weight per liter) were treated with monopolar rectangular pulses using a continuous flow system. The PEF energy required to achieve irreversible electropermeabilization was significantly reduced with the increase of the biomass concentration. Upon incubation of the permeabilized cells in water, only relatively small intracellular compounds were released. Release of 90% of the free amino acids and low molecular UV absorbing compounds, 80% of the glutathione, and ∼40% of the total phenol content was achieved about 2 h after pulsation and incubation of the suspensions at room temperature. At these conditions, the macromolecules (proteins and nucleic acids) were retained largely inside. Efficient protein release (∼90% from the total soluble protein) occurred only after dilution and incubation of the permeabilized cells in buffer with pH 8-9. Protein concentrates obtained by ultrafiltration (10 kDa cut off) had lower nucleic acid content (protein/nucleic acid ratio ∼100/4.5) in comparison with cell lysates obtained by mechanical disintegration. The obtained results allowed to conclude that PEF treatment can be used as an efficient alternative approach for production of yeast extracts with different composition, suitable for application in food, cosmetics and pharmaceutical industries.

Comparative effect of tenuazonic acid, diuron, bentazone, dibromothymoquinone and methyl viologen on the kinetics of Chl a fluorescence rise OJIP and the MR820 signal.

In this study, the comparative effect of TeA, DCMU, bentazone, DBMIB and MV on prompt fluorescence and the MR820 signal was simultaneously analyzed to provide an insight into how to elucidate their precise influence on Ageratina adenophora photosystems. The herbicides that interrupt electron transport beyond QA, such as TeA, DCMU and bentazone, mainly increased the J-step level of fluorescence rise kinetics as a result of accumulation of QA-, but showed differences in detail. The IP phase disappeared in the presence of DCMU and bentazone with a significant increase in FO value. TeA treatment retained the IP phase with lowering FM. As an inhibitor of plastoquinone re-oxidation, DBMIB increased the I-step (IP phase almost unnoticable) without changing FO and FM values. MV blocking PSI electron transfer through intercepting electrons from the FeS clusters suppressed the IP phase by decreasing the P level. Considering the WIP kinetics, TeA and DBMIB also affected PSI activity. After DCMU and MV treatment, the major change in the MR820 kinetics was the loss of the slow phase due to the complete prevention of electron movement from PSII to re-reduce PC+ and P700+. TeA, bentazone and DBMIB clearly suppressed the MR820 slow phase and decreased the re-reduction rate of PC+ and P700+ (Vred), significantly. However, there were still parts of electrons being donated to PC+ and P700+, showing a smaller slow phase and PC+ and P700+ re-reduction rate. Additionally, TeA and DBMIB also somewhat declined the fast phase and PC and P700 oxidation rate (Vox).

Delayed fluorescence in photosynthesis.

Photosynthesis is a very efficient photochemical process. Nevertheless, plants emit some of the absorbed energy as light quanta. This luminescence is emitted, predominantly, by excited chlorophyll a molecules in the light-harvesting antenna, associated with Photosystem II (PS II) reaction centers. The emission that occurs before the utilization of the excitation energy in the primary photochemical reaction is called prompt fluorescence. Light emission can also be observed from repopulated excited chlorophylls as a result of recombination of the charge pairs. In this case, some time-dependent redox reactions occur before the excitation of the chlorophyll. This delays the light emission and provides the name for this phenomenon-delayed fluorescence (DF), or delayed light emission (DLE). The DF intensity is a decreasing polyphasic function of the time after illumination, which reflects the kinetics of electron transport reactions both on the (electron) donor and the (electron) acceptor sides of PS II. Two main experimental approaches are used for DF measurements: (a) recording of the DF decay in the dark after a single turnover flash or after continuous light excitation and (b) recording of the DF intensity during light adaptation of the photosynthesizing samples (induction curves), following a period of darkness. In this paper we review historical data on DF research and recent advances in the understanding of the relation between the delayed fluorescence and specific reactions in PS II. An experimental method for simultaneous recording of the induction transients of prompt and delayed chlorophyll fluorescence and decay curves of DF in the millisecond time domain is discussed.

Application of a diffusion model to measure ion leakage of resurrection plant leaves undergoing desiccation.

Haberlea rhodopensis is a chlorophyll-retaining resurrection plant, which can survive desiccation to air dry state under both low light and sunny environments. Maintaining the integrity of the membrane during dehydration of resurrection plants is extremely important. In the present study, the diffusion model was improved and used for a first time to evaluate the changes in ion leakage through different cellular compartments upon desiccation of H. rhodopensis and to clarify the reasons for significant increase of electrolyte leakage from dry leaves. The applied diffusion approach allowed us to distinguish the performance of plants subjected to dehydration and subsequent rehydration under different light intensities. Well-hydrated (control) shade plants had lower and slower electrolyte leakage compared to control sun plants as revealed by lower values of phase amplitudes, lower rate constants and ion concentration. In well-hydrated and moderately dehydrated plants (50% relative water content, RWC) ion efflux was mainly due to leakage from apoplast. The electrolyte leakage sharply increased in severely desiccated leaves (8% RWC) from both sun and shade plants mainly due to ion efflux from symplast. After 1 day of rehydration the electrolyte leakage was close to control values, indicating fast recovery of plants. We suggest that the enhanced leakage in air-dried leaves should not be considered as damage but rather as a survival mechanism based on a reversible modification in the structure of cell wall, plasma membrane and alterations in vacuolar system of the cells. However, further studies should be conducted to investigate the changes in cell wall/plasma membrane to support this conclusion.

Structural and functional disorder in the photosynthetic apparatus of radish plants under magnesium deficiency.

Magnesium (Mg) is one of the significant macronutrients which is involved in the structural stabilisation of plant tissues and many enzymes such as PSII. The latter efficiency and performance were analysed, using chlorophyll (Chl) a fluorescence induction kinetics and microscopic images, to detect the changes in structure and function of photosynthetic apparatus of radish plants grown under Mg deficiency (Mgdef). Plants grown under Mgdef showed less PSII connectivity and fewer active primary electron acceptors (QA) oxidizing reaction centres than control plants. Confocal and electron microscopy analyses showed an increased amount of starch in chloroplasts, and 3,3'-diaminobenzidine (DAB)-uptake method revealed higher H2O2 accumulation under Mgdef. Prominent changes in the Chl a fluorescence parameters such as dissipated energy flux per reaction centre (DIo/RC), relative variable fluorescence at 150µs (Vl), and the sum of the partial driving forces for the events involved in OJIP fluorescence rise (DFabs) were observed under Mg deficiency. The latter also significantly affected some other parameters such as dissipated energy fluxes per cross-section (DIo/CSo), performance index for energy conservation from photons absorbed by PSII antenna until the reduction of PSI acceptors (PItotal), and relative variable fluorescence at 300µs (Vk). This work emphasises the use of chlorophyll fluorescence in combination with microscopic and statistical analyses to diagnose the effects of nutrients deficiency stress on plants at an early stage of its development as demonstrated for the example of Mgdef. Due to the short growth period and simple cultivation conditions of radish plant we recommend it as a new standard (model) plant to study nutrients deficiency and changes in plant photosynthetic efficiency under stress conditions.

Anthocyanin concentration depends on the counterbalance between its synthesis and degradation in plum fruit at high temperature.

Anthocyanin synthesis and degradation processes were analyzed at transcript, enzyme, and metabolite levels to clarify the effects of high temperature on the concentration of anthocyanin in plum fruit (Prunus salicina Lindl.). The transcript levels of PsPAL, PsCHS, and PsDFR decreased while those of PsANS and PsUFGT were similar at 35 °C compared with 20 °C. The activities of the enzymes encoded by these genes were all increased in fruits at 35 °C. The concentrations of anthocyanins were higher at 35 °C on day 5 but then decreased to lower values on day 9 compared with that at 20 °C. Furthermore, high temperature (35 °C) increased the concentration of hydrogen peroxide and the activity of class III peroxidase in the fruit. The concentration of procatechuic acid, a product of the reaction between anthocyanin and hydrogen peroxide, hardly changed at 20 °C but was significantly increased at 35 °C on day 9, indicating that anthocyanin was degraded by hydrogen peroxide, which was catalyzed by class III peroxidase. Based on mathematical modeling, it was estimated that more than 60-70% was enzymatically degraded on day 9 when the temperature increased from 20 °C to 35 °C. We conclude that at the high temperature, the anthocyanin content in plum fruit depend on the counterbalance between its synthesis and degradation.

Acclimatization of Photosynthetic Apparatus of Tor Grass (Brachypodium pinnatum) during Expansion.

The aim of this study was to understand the acclimatization mechanisms of photosynthetic apparatus in Brachypodium pinnatum (L.) P. Beauv grass during its expansion. Twelve populations differentiated by age: young (30-50 years old), intermediate age (ca. 100 y) and old (>300 y) were studied. It was confirmed that the decrease of the number of genotypes as a result of environmental stress and competition were reflected in changes in chlorophyll fluorescence (ChlF) parameters. The old stands were dominated by a few genotypes which seem to be the best acclimatized to the self-shading/competition by lowering their photosynthetic performance during light-phase of photosynthesis. On the other hand, the 'high-speed' photosynthetic rate observed in the young populations can be seen as acclimatization to very adverse conditions. Our results clearly confirm that ChlF is a powerful method of inferring physiological mechanisms of the expansion of tor grass. The Principal Component and Redundancy Analyses, followed with k-means classification, allowed to find the differentiation of groups of distinct ChlF parameters and enabled us to relate them to changes in genotypic diversity of populations. We conclude that the plastic morphological and physiological response to changeable habitat light conditions with its optimum in half-shade refers to its forest-steppe origin.

Thermotolerance of apple tree leaves probed by chlorophyll a fluorescence and modulated 820 nm reflection during seasonal shift.

During the seasonal shift from June to August, air temperatures increase. To explore how apple trees improve their thermotolerance during this shift, we examined the photochemical reaction capacity of apple tree leaves by simultaneous measurement of prompt chlorophyll fluorescence, delayed chlorophyll fluorescence, and modulated 820 nm reflection at varying temperatures. It was found that the reaction centers and antennae of photosystem II (PSII) and photosystem I (PSI), the donor side of PSII, the electron transfer capacity from QA to QB, and the reoxidation capacity of plastoquinol were all sensitive to heat stress, particularly in June. As the season shifted, apple tree leaves improved in thermotolerance. Interestingly, the acclimation to seasonal shift enhanced the thermotolerance of PSII and PSI reaction centers more than that of their antennae, and the activity of PSII more than that of PSI. This may be a strategy for plant adaptation to changes in environmental temperatures. In addition, results from prompt and delayed fluorescence, as well as modulated 820 nm reflection corroborate each other. We suggest that the simultaneous measurement of the three independent signals may provide more information on thermal acclimation mechanisms of photochemical reactions in plant leaves.

Photosynthetic performance during leaf expansion in Malus micromalus probed by chlorophyll a fluorescence and modulated 820nm reflection.

The simultaneous measurements of prompt chlorophyll a fluorescence, delayed chlorophyll a fluorescence and modulated 820nm reflection allow collection and correlation of complementary information for the three domains of the photosynthetic electron transport chain - the PSII electron donor side, electron transport between PSII and PSI, and the PSI electron acceptor side. In this study, we used this approach to investigate photochemical activity during Malus micromalus leaf expansion. The results showed that as leaves expanded, the antenna size per reaction center for the two systems became smaller, and the energetic connectivity of PSII units decreased gradually. Meanwhile, the light trapping efficiency of PSII, electron transfer capacity at the donor side of PSII, exchange capacity of PQs at the QB site and the reoxidation capacity of PQH2 were all increased as leaves expanded. However, the capacity of PQH2 reoxidation increased at a slower rate than the exchange capacity of PQs at the QB site. In general, during leaf development, the photochemical activity of both PSII and PSI increased, although the increase in PSII activity was faster relative to PSI. The results from the three independent signals corroborate each other.