How to Measure Grana - Ultrastructural Features of Thylakoid Membranes of Plant Chloroplasts.

Granum is a basic structural unit of the thylakoid membrane network of plant chloroplasts. It is composed of multiple flattened membranes forming a stacked arrangement of a cylindrical shape. Grana membranes are composed of lipids and tightly packed pigment-protein complexes whose primary role is the catalysis of photosynthetic light reactions. These membranes are highly dynamic structures capable of adapting to changing environmental conditions by fine-tuning photochemical efficiency, manifested by the structural reorganization of grana stacks. Due to a nanometer length scale of the structural granum features, the application of high-resolution electron microscopic techniques is essential for a detailed analysis of the granum architecture. This mini-review overviews recent approaches to quantitative grana structure analyses from electron microscopy data, highlighting the basic manual measurements and semi-automated workflows. We outline and define structural parameters used by different authors, for instance, granum height and diameter, thylakoid thickness, end-membrane length, Stacking Repeat Distance, and Granum Lateral Irregularity. This article also presents insights into efficient and effective measurements of grana stacks visualized on 2D micrographs. The information on how to correctly interpret obtained data, taking into account the 3D nature of grana stacks projected onto 2D space of electron micrograph, is also given. Grana ultrastructural observations reveal key features of this intriguing membrane arrangement, broadening our knowledge of the thylakoid network's remarkable plasticity.

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana.

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Compensation Mechanism of the Photosynthetic Apparatus in Arabidopsis thaliana ch1 Mutants.

The origin of chlorophyll b deficiency is a mutation (ch1) in chlorophyllide a oxygenase (CAO), the enzyme responsible for Chl b synthesis. Regulation of Chl b synthesis is essential for understanding the mechanism of plant acclimation to various conditions. Therefore, the main aim of this study was to find the strategy in plants for compensation of low chlorophyll content by characterizing and comparing the performance and spectral properties of the photosynthetic apparatus related to the lipid and protein composition in four selected Arabidopsis ch1 mutants and two Arabidopsis ecotypes. Mutation in different loci of the CAO gene, viz., NW41, ch1.1, ch1.2 and ch1.3, manifested itself in a distinct chlorina phenotype, pigment and photosynthetic protein composition. Changes in the CAO mRNA levels and chlorophyllide a (Chlide a) content in ecotypes and ch1 mutants indicated their significant role in the adjustment mechanism of the photosynthetic apparatus to low-light conditions. Exposure of mutants with a lower chlorophyll b content to short-term (1LL) and long-term low-light stress (10LL) enabled showing a shift in the structure of the PSI and PSII complexes via spectral analysis and the thylakoid composition studies. We demonstrated that both ecotypes, Col-1 and Ler-0, reacted to high-light (HL) conditions in a way remarkably resembling the response of ch1 mutants to normal (NL) conditions. We also presented possible ways of regulating the conversion of chlorophyll a to b depending on the type of light stress conditions.

Spatial Nano-Morphology of the Prolamellar Body in Etiolated Arabidopsis thaliana Plants With Disturbed Pigment and Polyprenol Composition.

The prolamellar body (PLB) is a periodic bicontinuous membrane structure based on tubular tetrahedral units. PLBs are present in plant etioplasts and, upon illumination, directly transform into the lamellar thylakoid networks within chloroplasts. Efficient tubular-lamellar rearrangement and later formation of the photosynthetically active thylakoid membranes are crucial steps in the development of plant autotrophy. PLB membranes are mainly composed of galactolipids, carotenoids, and protochlorophyllide (Pchlide), the chlorophyll precursor, bound in a complex with NADPH and Pchlide oxidoreductase. Although the PLB structure has been studied for over 50 years, the direct role of particular membrane components in the formation of the PLB paracrystalline network remains elusive. Moreover, despite the numerous literature data regarding the PLB geometry, their reliable comparative analysis is complicated due to variable experimental conditions. Therefore, we performed comprehensive ultrastructural and low-temperature fluorescence analysis of wild type Arabidopsis thaliana (Arabidopsis) seedlings grown in different conditions typical for studies on etiolated seedlings. We established that the addition of sucrose to the growing media significantly affected the size and compactness of the PLB. The etiolation period was also an important factor influencing the PLB structural parameters and the ratio of free to complex-bound Pchlide. Thus, a reliable PLB structural and spectral analysis requires particular attention to the applied experimental conditions. We investigated the influence of the pigment and polyprenol components of the etioplast membranes on the formation of the PLB spatial structure. The PLB 3D structure in several Arabidopsis mutants (ccr1-1, lut5-1, szl1-1npq1-2, aba1-6, pif1, cpt7) with disturbed levels of particular pigments and polyprenols using electron tomography technique was studied. We found that the PLB nano-morphology was mainly affected in the pif1 and aba1-6 mutants. An increased level of Pchlide (pif1) resulted in the substantial shift of the structural balance between outer and inner PLB water channels and overall PLB compactness compared to wild type plants. The decrease in the relative content of ß-branch xanthophylls in aba1-6 plants was manifested by local disturbances in the paracrystalline structure of the PLB network. Therefore, proper levels of particular etioplast pigments are essential for the formation of stable and regular PLB structure.

Specific Composition of Lipid Phases Allows Retaining an Optimal Thylakoid Membrane Fluidity in Plant Response to Low-Temperature Treatment.

Thylakoid membranes isolated from leaves of two plant species, the chilling tolerant (CT) pea and chilling sensitive (CS) runner bean, were assessed for the composition of lipids, carotenoids as well as for the arrangement of photosynthetic complexes. The response to stress conditions was investigated in dark-chilled and subsequently photo-activated detached leaves of pea and bean. Thylakoids of both species have a similar level of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), but different sulfoquinovosyldiacylglycerol to phosphatidylglycerol (PG) ratio. In pea thylakoid fraction, the MGDG, DGDG and PG, have a higher double bond index (DBI), whereas bean thylakoids contain higher levels of high melting point PG. Furthermore, the lutein to the ß-carotene ratio is higher in bean thylakoids. Smaller protein/lipid ratio in pea than in bean thylakoids suggests different lipid-protein interactions in both species. The differences between species are also reflected by the course of temperature-dependent plots of chlorophyll fluorescence pointing various temperatures of the lipid phase transitions of pea and bean thylakoids. Our results showed higher fluidity of the thylakoid membrane network in pea than in bean in optimal temperature conditions. Dark-chilling decreases the photochemical activity and induces significant degradation of MGDG in bean but not in pea leaves. Similarly, substantial changes in the arrangement of photosynthetic complexes with increase in LHCII phosphorylation and disturbances of the thylakoid structure take place in bean thylakoids only. Changes in the physical properties of bean thylakoids are manifested by the conversion of a three-phase temperature-dependent plot to a one-phase plot. Subsequent photo-activation of chilled bean leaves caused a partial restoration of the photochemistry and of membrane physical properties, but not of the photosynthetic complexes arrangement nor the thylakoid network structure. Summarizing, the composition of the thylakoid lipid matrix of CT pea allows retaining the optimal fluidity of its chloroplast membranes under low temperatures. In contrast, the fluidity of CS bean thylakoids is drastically changed, leading to the reorganization of the supramolecular structure of the photosynthetic complexes and finally results in structural remodeling of the CS bean thylakoid network.

Galactolipid deficiency disturbs spatial arrangement of the thylakoid network in Arabidopsis thaliana plants.

The chloroplast thylakoid network is a dynamic structure which, through possible rearrangements, plays a crucial role in regulation of photosynthesis. Although the importance of the main components of the thylakoid membrane matrix, galactolipids, in the formation of the network of internal plastid membrane was found before, the structural role of monogalactosyldiacylglycerol (MGDG) and digalactosylidacylglycerol (DGDG) is still largely unknown. We elucidated detailed structural modifications of the thylakoid membrane system in Arabidopsis thaliana MGDG- and DGDG-deficient mutants. An altered MGDG/DGDG ratio was structurally reflected by formation of smaller grana, local changes in grana stacking repeat distance, and significant changes in the spatial organization of the thylakoid network compared with wild-type plants. The decrease of the MGDG level impaired the formation of the typical helical grana structure and resulted in a 'helical-dichotomic' arrangement. DGDG deficiency did not affect spatial grana organization but changed the shape of the thylakoid membrane network in situ from lens like into a flattened shape. Such structural disturbances were accompanied by altered composition of carotenoid and chlorophyll-protein complexes, which eventually led to the decreased photosynthetic efficiency of MGDG- and DGDG-deficient plants.

Dark-chilling and subsequent photo-activation modulate expression and induce reversible association of chloroplast lipoxygenase with thylakoid membrane in runner bean (Phaseolus coccineus L.).

Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases that catalyse oxygenation of polyunsaturated fatty acids. This reaction is the first step in biosynthesis of oxylipins, which play important and diverse roles in stress response. In this study, we identified four LOX genes (PcLOXA, B, C, D) in chilling-sensitive runner bean (Phaseolus coccineus L.) plant and analyzed their expression patterns during long term dark-chilling (4 °C) stress and during day/night (21ºC/4 °C) temperature fluctuations. Three of the four identified LOX genes, namely PcLOXA, PcLOXB and PcLOXD, were induced by wounding stress, while only the PcLOXA was induced by dark-chilling of both detached (wounded) leaves and whole plants. We identified PcLOXA as a chloroplast-targeted LOX protein and investigated its expression during chilling stress in terms of abundance, localization inside chloroplasts and interactions with the thylakoid membranes. The analysis by immunogold electron microscopy has shown that more than 60% of detectable PcLOXA protein was associated with thylakoids, and dark-chilling of leaves resulted in increased amounts of this protein detected within grana margins of thylakoids. This effect was reversible under subsequent photo-activation of chilled leaves. PcLOXA binding to thylakoids is not mediated by the posttranslational modification but rather is based on direct interactions of the protein with membrane lipids; the binding strength increases under dark-chilling conditions.

Dark-chilling induces substantial structural changes and modifies galactolipid and carotenoid composition during chloroplast biogenesis in cucumber (Cucumis sativus L.) cotyledons.

Plants in a temperate climate are often subject to different environmental factors, chilling stress among them, which influence the growth especially during early stages of plant development. Chloroplasts are one of the first organelles affected by the chilling stress. Therefore the proper biogenesis of chloroplasts in early stages of plant growth is crucial for undertaking the photosynthetic activity. In this paper, the analysis of the cotyledon chloroplast biogenesis at different levels of plastid organization was performed in cucumber, one of the most popular chilling sensitive crops. Influence of low temperature on the ultrastructure was manifested by partial recrystallization of the prolamellar body, the formation of elongated grana thylakoids and a change of the prolamellar body structure from the compacted "closed" type to a more loose "open" type. Structural changes are strongly correlated with galactolipid and carotenoid content. Substantial changes in the galactolipid and the carotenoid composition in dark-chilled plants, especially a decrease of the monogalactosyldiacylglycerol to digalactosyldiacylglycerol ratio (MGDG/DGDG) and an increased level of lutein, responsible for a decrease in membrane fluidity, were registered together with a slower adaptation to higher light intensity and an increased level of non-photochemical reactions. Changes in the grana thylakoid fluidity, of their structure and photosynthetic efficiency in developing chloroplasts of dark-chilled plants, without significant changes in the PSI/PSII ratio, could distort the balance of photosystem rearrangements and be one of the reasons of cucumber sensitivity to chilling.

Overlapping toxic effect of long term thallium exposure on white mustard (Sinapis alba L.) photosynthetic activity.

BACKGROUND: Heavy metal exposure affect plant productivity by interfering, directly and indirectly, with photosynthetic reactions. The toxic effect of heavy metals on photosynthetic reactions has been reported in wide-ranging studies, however there is paucity of data in the literature concerning thallium (Tl) toxicity. Thallium is ubiquitous natural trace element and is considered the most toxic of heavy metals; however, some plant species, such as white mustard (Sinapis alba L.) are able to accumulate thallium at very high concentrations. In this study we identified the main sites of the photosynthetic process inhibited either directly or indirectly by thallium, and elucidated possible detoxification mechanisms in S. alba. RESULTS: We studied the toxicity of thallium in white mustard (S. alba) growing plants and demonstrated that tolerance of plants to thallium (the root test) decreased with the increasing Tl(I) ions concentration in culture media. The root growth of plants exposed to Tl at 100 µg L(-1) for 4 weeks was similar to that in control plants, while in plants grown with Tl at 1,000 µg L(-1) root growth was strongly inhibited. In leaves, toxic effect became gradually visible in response to increasing concentration of Tl (100 - 1,000 µg L(-1)) with discoloration spreading around main vascular bundles of the leaf blade; whereas leaf margins remained green. Subsequent structural analyses using chlorophyll fluorescence, microscopy, and pigment and protein analysis have revealed different effects of varying Tl concentrations on leaf tissue. At lower concentration partial rearrangement of the photosynthetic complexes was observed without significant changes in the chloroplast structure and the pigment and protein levels. At higher concentrations, the decrease of PSI and PSII quantum yields and massive oxidation of pigments was observed in discolored leaf areas, which contained high amount of Tl. Substantial decline of the photosystem core proteins and disorder of the photosynthetic complexes were responsible for disappearance of the chloroplast grana. CONCLUSIONS: Based on the presented results we postulate two phases of thallium toxicity on photosynthesis: the non-destructive phase at early stages of toxicant accumulation and the destructive phase that is restricted to the discolored leaf areas containing high toxicant content. There was no distinct border between the two phases of thallium toxicity in leaves and the degree of toxicity was proportional to the migration rate of the toxicant outside the vascular bundles. The three-fold (nearly linear) increase of Tl(I) concentration was observed in damaged tissue and the damage appears to be associated with the presence of the oxidized form of thallium - Tl(III).

Three-Dimensional Visualization of the Tubular-Lamellar Transformation of the Internal Plastid Membrane Network during Runner Bean Chloroplast Biogenesis.

Chloroplast biogenesis is a complex process that is integrated with plant development, leading to fully differentiated and functionally mature plastids. In this work, we used electron tomography and confocal microscopy to reconstruct the process of structural membrane transformation during the etioplast-to-chloroplast transition in runner bean (Phaseolus coccineus). During chloroplast development, the regular tubular network of paracrystalline prolamellar bodies (PLBs) and the flattened porous membranes of prothylakoids develop into the chloroplast thylakoids. Three-dimensional reconstruction is required to provide us with a more complete understanding of this transformation. We provide spatial models of the bean chloroplast biogenesis that allow such reconstruction of the internal membranes of the developing chloroplast and visualize the transformation from the tubular arrangement to the linear system of parallel lamellae. We prove that the tubular structure of the PLB transforms directly to flat slats, without dispersion to vesicles. We demonstrate that the grana/stroma thylakoid connections have a helical character starting from the early stages of appressed membrane formation. Moreover, we point out the importance of particular chlorophyll-protein complex components in the membrane stacking during the biogenesis. The main stages of chloroplast internal membrane biogenesis are presented in a movie that shows the time development of the chloroplast biogenesis as a dynamic model of this process.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.

Chloroplast biogenesis - correlation between structure and function.

Chloroplast biogenesis is a multistage process leading to fully differentiated and functionally mature plastids. Complex analysis of chloroplast biogenesis was performed on the structural and functional level of its organization during the photoperiodic plant growth after initial growth of seedlings in the darkness. We correlated, at the same time intervals, the structure of etioplasts transforming into mature chloroplasts with the changes in the photosynthetic protein levels (selected core and antenna proteins of PSI and PSII) and with the function of the photosynthetic apparatus in two plant species: bean (Phaseolus vulgaris L.) and pea (Pisum sativum L). We selected these plant species since we demonstrated previously that the mature chloroplasts differ in the thylakoid organization. We showed that the protein biosynthesis as well as photosynthetic complexes formation proceeds gradually in both plants in spite of periods of darkness. We found that both steady structural differentiation of the bean chloroplast and reformation of prolamellar bodies in pea were accompanied by a gradual increase of the photochemical activity in both species. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes.

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.