Structure and Function of Chloroplast Membrane II. The Effects of Potassium and Magnesium Ions on the Absorption Spectrum and Photosystem II Function of Two Types of Chloroplast Me-mbranes

After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr., we obtained an incompletely developed chloroplast membrane. It was then compared with a completely developed chloroplast membrane obtaining from wheat seedlings grown under normal light-dark regime. We investgated the effect of various cations and their concentrations on the absorption spectrum and the photosystem Ⅱ function of the above two types of chloroplast membranes. A similar effect of potassium and magnesium ions on the absorption spectra of completely developed chloroplast membrane was observed. They decreased the absorption peak values at both the red and blue regions of the chloroplast membrane in the same manner. The degree of decrease in the peak value is proportional to ion concentration. But in the incompletely developed chloroplast membranes similar phenomenon was not observed. In the presence of K+ and Mg2+ of various concentrations, the absorptionn peaks at the red region overlapped almost completely, and these at the blue region only changed slightly with ion concentrations. DCIP photoreduction rate of the two types of chloroplast membranes was stimulated by the addition of K+ and Mg2+ in various concentrations. But the degree of stimulation in the two types of membranes was quite different. In the presence of l00 mM KCl or 5.0 mM MgCl2, DCIP photoreduction rate of completely developed chloroplast membranes was enhanced by 76.8% and 68.9% respectively, whereas in incompletely developed chloroplast membranes it was only increased by 56.3% and 36.4% respectively. The causes of the effects of cations on the absorption spectrum and the photosystem Ⅱ function of two types of chloroplast membranes were discussed.     

Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.     

Structure and Function of Chloroplast Membrane XI. The Effects of Linolenic Acid on the Structure and the Absorption and Fluorescence Spectra of Wheat Chloroplast Membranes as well as Regulations by MgCl2

The effects of various concentrations of linolenic acid on the structure and the absorption and fluorescence spectra of wheat chloroplast membranes were studied. Linolenic acid increases the absorption peaks in both red and blue regions of the chloroplast membranes. The degree of increase in the absorption peaks is proportional to the concentration within a range of concentration. Linolenic acid increased the fluorescence yield of F685 and F738 of chloroplast membranes. Electron microscopical studies revealed that the increases were mainly due to the disappearance of grana stacks and the unfolding of thylakoid membranes. The causes of effects of linolenic acid on the absorption and fluorescence spectra and the structure of chloroplast membranes as well as the reversion and regulation by MgCl2 were discussed.     

Dynamic flexibility in the structure and function of photosystem II in higher plant thylakoid membranes: the grana enigma

Grana are not essential for photosynthesis, yet they are ubiquitous in higher plants and in the recently evolved Charaphyta algae; hence grana role and its need is still an intriguing enigma. This article discusses how the grana provide integrated and multifaceted functional advantages, by facilitating mechanisms that fine-tune the dynamics of the photosynthetic apparatus, with particular implications for photosystem II (PSII). This dynamic flexibility of photosynthetic membranes is advantageous in plants responding to ever-changing environmental conditions, from darkness or limiting light to saturating light and sustained or intermittent high light. The thylakoid dynamics are brought about by structural and organizational changes at the level of the overall height and number of granal stacks per chloroplast, molecular dynamics within the membrane itself, the partition gap between appressed membranes within stacks, the aqueous lumen encased by the continuous thylakoid membrane network, and even the stroma bathing the thylakoids. The structural and organizational changes of grana stacks in turn are driven by physicochemical forces, including entropy, at work in the chloroplast. In response to light, attractive van der Waals interactions and screening of electrostatic repulsion between appressed grana thylakoids across the partition gap and most probably direct protein interactions across the granal lumen (PSII extrinsic proteins OEEp-OEEp, particularly PsbQ-PsbQ) contribute to the integrity of grana stacks. We propose that both the light-induced contraction of the partition gap and the granal lumen elicit maximisation of entropy in the chloroplast stroma, thereby enhancing carbon fixation and chloroplast protein synthesizing capacity. This spatiotemporal dynamic flexibility in the structure and function of active and inactive PSIIs within grana stacks in higher plant chloroplasts is vital for the optimization of photosynthesis under a wide range of environmental and developmental conditions.     

不同叶色水稻叶绿体密度及基粒结构的计算机图象分析

水稻叶绿体计算机图象分析表明,随着叶片色级的提高,叶绿体表面积密度、体积密度以及两者的比值都相应增加。深色稻叶基粒堆直径与高度、类囊体垛叠数与类囊体厚度、叶绿素与类胡萝卜素含量、气孔导度与净光合率均大于浅色叶片。深色叶片基粒堆密集,有些基粒类囊体出现沿叶绿体长轴方向排列整齐现象;浅色叶片基粒堆稀疏,其中较大的基粒类囊体与长轴呈倾斜排列。     

Immunogold localization of LHC II proteins in chloroplasts with different degrees of grana stacking induced by SAN-9789.

The amount and distribution of proteins of the light-harvesting complex associated with photosystem II (PS II) were investigated using immunogold labelling of chloroplasts of wheat ( Triticum aestivum L. cv. Walde). The seedlings were grown in weak red light (16 mW m⁻²) after imbibition of grains with SAN-9789 (Norflurazon, 0.028 to 28 mg I⁻¹). Chloroplasts of these plants exhibited thylakoids with different degrees of stacking. Thylakoids of untreated plants grown in a greenhouse had most gold particles per unit membrane length in both appressed and non-appressed regions compared to red light grown plants. The ratios of labelling between appressed and non-appressed membranes were fairly constant in red light- and greenhouse-grown plants. The labelling densities were 2.5–3 times higher in the appressed thylakoids compared to the non-appressed thylakoids. However, at a SAN concentration of 2.8 mg I⁻¹ there was a sharp decrease in thylakoid appressions and in labelling density of both appressed and non-appressed membranes. The total amount of particles per chloroplast was also much lower as compared to that at lower SAN concentrations. Plants treated with the highest concentration of SAN (28 mg I⁻¹) contained chloroplasts devoid of normal grana structures. In these plastids, the thylakoids were elongated and single. The labelling density in these membranes was ca 50% of that observed at 2.8 mg I⁻¹. This paper thus supports earlier observations that proteins of the light-harvesting complex of PS II (LHC II) are mainly localized in the appressed regions of the grana membranes, and may be involved in the formation of grana.     

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg²⁺, to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.     

Arrangement of Photosystem II and ATP Synthase in Chloroplast Membranes of Spinach and Pea

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

Structural and Immunological Characterization of the Cuscuta pentagona L. Chloroplast

Structural and immunochemical studies were used to determinethe photosynthetic potential of the dodder (Cuscuta pentagona)chloroplast. Ultrastructural studies revealed that thylakoidmembranes of pre-parasitic phase Cuscuta pentagona are almostall organized into long, overlapping grana stacks of mainlytwo to five thylakoids with little space between adjacent stacks.Immunoblots reveal chloroplast proteins associated with PSIand II, as well as cytochrome f and plastocyanin. Stromal extractscontained immmunologically-detectable RuBisCO and phosphoribulokinase.Cytochemical localizations of the oxidizing side of PSI showedproduct localization on the lumen side of the thylakoid. Immunocytochemicallocalizations of RuBisCO reveal exclusive labeling in the stroma,whereas antibodies to the PSII proteins, light-harvesting Chla/b complex and the oxygen-evolving complex of PSII, are concentratedover the thylakoids. A limited capacity for CO₂ fixation wasfound in seedlings by monitoring CO₂ exchange rates in the presenceand absence of atrazine. These data indicate that the chloroplastfrom this species of dodder contains a number of the proteinsrequired for a successful fixation of CO₂ and the proteins inthe thylakoids are organized much like other higher plants,with the exception of the large percentage of the thylakoidsorganized into grana. (Received August 10, 1998; Accepted April 3, 1999)     

Reorganization of the chloroplast thylakoid system during decay of acquired thermotolerance of photosynthesis and aging of wheat leaves

A 3 hours heating at 39 degrees C of 14-day old wheat plants increases the termotolerance of photosynthesis, and also the length and number of thylakoids in chloroplast in mature leaves. The acquired termotolerance disappears within 10 days. Simultaneously the intensity of photosynthesis and the length of thylakoids decrease. Reduction of photosynthesis ability and of thylakoid membranes occurs in the first leaves of non-hardened plants during 14-29 days after sowing. The intensity of photosynthesis in plants of both variants positively correlates with the length of grana membranes and with the total length of membranes of all thylakoids. Besides, a positive correlation was detected between the intensity of photosynthesis and the share of small (2-7 thylakoids) grana and the length of their membranes in non-hardened plants. The level of thermotolerance of photosynthesis in leaves in heat hardened plants correlates positively with the length of grana membranes and with the total length of all thylakoid membranes and the share of small grana.     

Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.     

Effect of Doubled-CO2 Concentration on the Ultrastructure of Chloroplasts from Medicago sativa and Setaria italica

It has been reported in quite a number of literatures that doubled CO2 concentration increased the photosynthetic rate and dry matter production of C3 plants, but substantially affected C4 plants little. However, why may CO2 enrichment promote growth and either no change or decrease reproductive allocation of the C3 species, but havinag no effects on growth characteristics of the C4 plants? So far, there has been no satisfactory explanation on that mentioned above, except the differences in their CO2 compensatory points. In the past, although some studies on ultrastructure of the chloroplasts under doubled CO2 concentration were limitedly conducted. Almost all the relevant experimental materials were only from C3 plants not from C4 plants, and even though the results were of inconsistancy. Thereby, it needs to verify whether the differences in photosynthesis of C3 and C4 plants at doubled CO2 level is caused by the difference in their chloroplast deterioration. Experiments to this subject were conducted at the Botanical Garden of Institute of Botany, Academia Sinica in 1993 and 1994. Both experimental materials from C3 plant alfalfa (Medicago sativa) and C4 plant foxtail millet (Setaria italica) were cultivated in the cylindrical open-top chambers (2.2 m in diameter × 2.4 m in height) with aluminum frames covered by polyethylene film. Natural air or air with 350× 10-6 CO2 were blown from the bottom of the chamber space with constant temperature between inside and outside of the chamber 〈0.2℃〉. Electron microscopic observation revealed that the ultrastructure of the chloroplasts from C3 plant Medicago sativa and C4 plant Seteria italica growing under the same doubled CO2 concentration were quite different from each other. The differential characteristics in ultrastructure of chloro plasts displayed mainly in the configuration of thylakoid membrances and the accumulation of starch grains. They were as follows: 1. The most striking feature was the building up of starch grains in the chloroplasts of the bundle sheath cells (BSCs) and the mesophyll cells (MCs) at doubled CO2 concentra tion. The starch grains appeared centrifugally first in the BSCs and then in the chloroplast of the other MCs. It was worthy to note that the starch grains in the chloroplasts of C4 plant Setaria ira/ica were much more than those of the C3 plant Medicago sativa . The decline of photosynthesis in the doubled CO2-grown C4 plants might be caused by an over accumulation of starch grains, that deformed the chloroplast even demaged the stroma thylakoids and grana. There might exsist a correlation between the comformation of thylakoid system and starch grain accumulation, namely conversion and transfer of starch need energy from ATP, and coupling factor (CF) for ATP formation distributed mainly on protoplastic surface (PSu) of stroma thylakoid membranes, as well as end and margin membranes of grana thylakoids. Thereby, these results could provide a conclusive evidence for the reason of non effectiveness on growth characteristics of C4 plant. 2. Under normal condition , the mature chlolroplats of higher plants usually develop complete and regularly arranged photosynthetic membrane systems . Chloroplasts from the C4 plant Setaria italica, however, exerted significant changes on stacking degree, grana width and stroma thylakoid length under doubled CO2 concentration; In these changes, the grana stacks were smaller and more numerous, and the number of thylakoids per granum was greatly increased, and the stroma thylakoid was greatly lengthened as compared to those of the control chloroplasts. But the grana were mutually intertwined by stroma thylakoid. The integrity of some of the grana were damaged due to the augmentation of the intrathylakoid space . Similarly, the stroma thylakoids were also expanded. In case. the plant was seriously effected by doubled CO2 concentration as observed in C4 plant Setaria italica , its chloroplasts contained merely the stroma (matrix) with abundant starch grains, while grana and stroma thylakoid membranes were unrecognizable, or occasionally a few residuous pieces of thylakoid membranes could be visualized, leaving a situation which appeared likely to be chloroplast deterioration. However, under the same condition the C3 plant Medicago sativa possessed normally developed chloroplasts, with intact grana and stroma thylakoid membranes. Its chloroplasts contained grana intertwined with stroma thylakoid membranes, and increased in stacking degree and granum width, in spite of more accumulated starch grains within the chloroplasts. These configuration changes of the thylakoid system were in consistant with the results of the authors another study on chloroplast function, viz. the increased capacity of chloroplasts for light absorption and efficiency of PSⅡ.     

The constant proportion of grana and stroma lamellae in plant chloroplasts

The relative proportion of stroma lamellae and grana end membranes was determined from electron micrographs of 58 chloroplasts from 21 different plant species. The percentage of grana end membranes varied between 1 and 21% of the total thylakoid membrane indicating a large variation in the size of grana stacks. By contrast the stroma lamellae account for 20.3 ± 2.5 ( sd )% of the total thylakoid membrane. A plot of percentage stroma lamellae against percentage of grana end membranes fits a straight line with a slope of zero showing that the proportion of stroma lamellae is independent of the size of the grana stacks. That stroma lamellae account for about 20% of the thylakoid membrane is in agreement with fragmentation and separation analysis (Gadjieva et al . Biochim. Biophys. Acta 144: 92–100, 1999). Chloroplasts from spinach, grown under high or low light, were fragmented by sonication and separated by countercurrent distribution into two vesicle populations originating from grana and stroma lamellae plus end membranes, respectively. The separation diagrams were very similar lending independent support for the notion that the proportion of stroma lamellae is constant. The results are discussed in relation to the composition and function of the chloroplast in plants grown under different environmental conditions, and in relation to a recent quantitative model for the thylakoid (Albertsson, Trends Plant Sci. 6: 349–354, 2001).     

Light-induced structural changes of thylakoids in normal and carotenoid deficient chloroplasts of maize

Summary Changes of membrane thickness and loculi were studied after red (650 nm) and far-red (707 nm) light in thylakoids of maize with different stacking and pigment compositions.The most intensive shrinkage of thylakoid membranes occurred in grana and under red light. Membranes of stroma thylakoids responded more to far-red light. Bundle sheath thylakoid membranes did not change in thickness. Loculi decreased in all types of thylakoids under both, red and far-red light. Thylakoids obtained from a -carotenic mutant exhibited a contrasting response: swelling under red light followed by photodestruction. Changes under far-red light were similar to that of normal stroma thylakoids.The data on normal chloroplasts show that the light induced shrinkage of membranes and the decrease of loculi are coupled to a different degree in various kinds of thylakoids; that the thylakoid flattening can be correlated with the Photosystem content of the membranes; and that two kinds of single thylakoids (stroma lamellae and bundle sheath lamellae) are different in molecular structure and function.Data on carotenoid deficient chloroplasts indicate a photooxidative destruction of the thylakoids by Photosystem 2 that occurs in the absence of normal carotenoids.     

The importance of grana stacking for xanthophyll cycle-dependent NPQ in the thylakoid membranes of higher plants

In the present study we have examined the effects of grana stacking on the rate of violaxanthin (Vx) de-epoxidation and the extent of non-photochemical quenching of chlorophyll a fluorescence (NPQ) in isolated thylakoid membranes of spinach. Our results show that partial and complete unstacking of thylakoids in reaction media devoid of sorbitol and MgCl₂ did not significantly affect the efficiency of Vx de-epoxidation. Under high light (HL) illumination we found slightly higher values of Vx conversion in stacked membranes, whereas in thylakoids incubated at pH 5.2 in the dark, representing the pH-optimum of Vx de-epoxidase, de-epoxidation was slightly increased in the unstacked membranes. Partial and complete unstacking of grana membranes, however, had a dramatic effect on the HL-induced NPQ. High NPQ values could only be achieved in stacked thylakoid membranes in the presence of MgCl₂ and sorbitol. In unstacked membranes NPQ was drastically decreased. The effects of grana stacking on the xanthophyll cycle-dependent component of NPQ were even more pronounced, and complete unstacking of thylakoid membranes led to a total loss of this quenching component. Our data imply that grana stacking in the thylakoid membranes of higher plants is of high importance for the process of overall NPQ. For the xanthophyll cycle-dependent component of NPQ it may even be essential. Possible effects of grana stacking on the mechanism of zeaxanthin-dependent quenching are discussed.     

Significance of structural variation in thylakoid membranes in maintaining functional photosystems during reproductive growth

Structural variation in the stroma‐grana (SG) arrangement of the thylakoid membranes, such as changes in the thickness of the grana stacks and in the ratio between grana and inter‐grana thylakoid, is often observed. Broadly, such alterations are considered acclimation to changes in growth and the environment. However, the relation of thylakoid morphology to plant growth and photosynthesis remains obscure. Here, we report changes in the thylakoid during leaf development under a fixed light condition. Histological studies on the chloroplasts of fresh green Arabidopsis leaves have shown that characteristically shaped thylakoid membranes lacking the inter‐grana region, referred to hereafter as isolated‐grana (IG), occurred adjacent to highly ordered, large grana layers. This morphology was restored to conventional SG thylakoid membranes with the removal of bolting stems from reproductive plants. Statistical analysis showed a negative correlation between the incidences of IG‐type chloroplasts in mesophyll cells and the rates of leaf growth. Fluorescence parameters calculated from pulse‐amplitude modulated fluorometry measurements and CO₂ assimilation data showed that the IG thylakoids had a photosynthetic ability that was equivalent to that of the SG thylakoids under moderate light. However, clear differences were observed in the chlorophyll a/b ratio. The IG thylakoids were apparently an acclimated phenotype to the internal condition of source leaves. The idea is supported by the fact that the life span of the IG thylakoids increased significantly in the later developing leaves. In conclusion, the heterogeneous state of thylakoid membranes is likely important in maintaining photosynthesis during the reproductive phase of growth.     

Light-Induced Chloroplast Differentiation in Soybean Cells in Suspension Culture : Ultrastructural Changes during the Bleaching and Greening Cycles

Suspension cultures of SB-P cells of soybean (Glycine max) provide a novel, reproducible, and readily manipulable greening system useful for inducing chloroplast differentiation. The cells are subcultured and grown heterotrophically (3% sucrose) in the dark for at least three successive 14-day periods, subcultured and grown in the dark for 7 days more, and finally placed under white light and grown photoautotrophically. Chlorophyll begins to accumulate by 1 hour of light and continues up to 12 days. The chlorophyll a:chlorophyll b ratio is 3:1. Dark-grown cells contain a small amount of total carotenoids which increase 10-fold during greening. Chloroplast differentiation is strictly light dependent, with photosynthetic pigments accumulating in the light and being lost from cells returned to the dark. In the dark, the chloroplasts dedifferentiate to amyloplasts as the organized thylakoid network is lost and starch accumulates. Under continuous light, the amyloplasts differentiate into mature chloroplasts as the organelle elongates, becomes spanned by several bands of thylakoids, and undergoes grana formation. Chloroplast differentiation in SB-P cells is similar to that in intact angiosperms developing under normal light-dark cycles.     

Study of membrane-bound ribosomes during pea chloroplasts formation]

Membrane-bound ribosomes of chloroplasts, isolated from pea seedlings during grana formation, can be partially liberated by 0.5 M KCl and 0.001 M puromycin. In case of mature chloroplasts, after the completion of grana formation process these agents are inefficient, and liberation of ribosomes and polyribosomes may be achieved only after solubilization of thylakoid membranes by 1% Triton X-100. Electron microscopic study of the heavy membrane fraction of young chloroplasts reveals electron-transparent membranes, containing rings and discs of thylakoids with a diameter of about 2 mum. These rings are liberated together with ribosomes under the action of 0.5 M KCl; Triton X-100 liberates equally-sized annular polyribosomes. The rings detected in chloroplast membranes at early stages of development are regarded as structures, precursor grana thylakoids, and the annular polyribosomes included into them as immediate participants of thylakoid morphogenesis.