Mechanistic aspects of the xanthophyll dynamics in higher plant thylakoids

Plant thylakoids have a highly conserved xanthophyll composition, consisting of β-carotene, lutein, neoxanthin and a pool of violaxanthin that can be converted to antheraxanthin and zeaxanthin in excess light conditions. Recent work has shown that xanthophylls undergo dynamic changes, not only in their composition but also in their distribution among Lhc proteins. Xanthophylls are released from specific binding site in the major trimeric LHCII complex of photosystem II and are subsequently bound to different sites into monomeric Lhcb proteins and dimeric Lhca proteins. In this work we review available evidence from in vivo and in vitro studies on the structural determinants that control xanthophyll exchange in Lhc proteins. We conclude that the xanthophyll exchange rate is determined by the structure of individual Lhc gene products and it is specifically controlled by the lumenal pH independently from the activation state of the violaxanthin de-epoxidase enzyme. The xanthophyll exchange induces important modifications in the organization of the antenna system of Photosystem II and, possibly of Photosystem I. Major changes consist into a modulation of the light harvesting efficiency and an increase of the protection from lipid peroxidation. The xanthophyll cycle thus appears to be a signal transduction system for co-ordinated regulation of the photoprotection mechanisms under persistent stress from excess light.     

Further studies of the relationship between cation-induced chlorophyll fluorescence and thylakoid membrane stacking changes

Salt-induced changes in thylakoid stacking and chlorophyll fluorescence do not occur with granal membranes obtained by treatment of stacked thylakoids with digitonin. In contrast to normal untreated thylakoids, digitonin prepared granal membranes remain stacked under all ionic conditions and exhibit a constant high level of chlorophyll fluorescence. However, unstacking of these granal membranes is possible if they are pretreated with either acetic anhydride or linolenic acid.Trypsin treatment of the thylakoids inhibits the salt induced chlorophyll fluorescence and stacking changes but stacking of these treated membranes does occur when the pH is lowered, with the optimum being at about pH 4.5. This type of stacking is due to charge neutralization and does not require the presence of the 2000 dalton fragment of the polypeptide associated with the $\text{chlorophyll}\text{a}\text{chlorophyll}\text{b}$ light harvesting complex and known to be lost during treatment with trypsin (Mullet, J.E. and Arntzen, C.J. (1980) Biochim. Biophys. Acta 589, 100–117).Using the method of 9-aminoacridine fluorescence quenching it is argued that the surface charge density, on a chlorophyll basis, of unstacked thylakoid membranes is intermediate between digitonin derived granal and stromal membranes, with granal having the lowest value.The results are discussed in terms of the importance of surface negative charges in controlling salt induced chlorophyll fluorescence and thylakoid stacking changes. In particular, emphasis is placed on a model involving lateral diffusion of different types of chlorophyll protein complex within the thylakoid lipid matrix.     

Segregation of the photosystems in higher plant thylakoids and short- and long-term regulation by a mesoscopic approach

In this paper we consider the relationship between the lateral segregation of photosystems I and II in the grana and characteristics of the short- and long-term regulation in thylakoids following the mesoscopic approach. Our study is thermodynamic; it is based on the Flory-Huggins theory for binary mixtures and the McMillan-Mayer theory of heterogeneous solutions. We demonstrate that state transitions promote rearrangement of photosystems by either favoring their mixing after LHCII phosphorylation or enhancing their segregation after LHCII dephosphorylation. This regulation influences the entire system properties locally. We also demonstrate that the variations of the photosystem ratio promote rearrangement of the photosystems preserving their segregation. This regulation influences the entire system properties globally. The studies presented are another indication of the importance of the segregation of the photosystems in the grana thylakoids of higher plants. Segregation of PSIs and PSIIs is a signature of the spinodal decomposition, which is a fine regulatory mechanism, related to both the short- and long-term adaptations of the photosynthetic apparatus in higher plant thylakoids.     

Protein dynamics in thylakoids of the desiccation-tolerant plant Boea hygroscopica during dehydration and rehydration

Plants of Boea hygroscopica F. Muell were dehydrated to 9% relative water content (RWC) by withholding water for 26 d, and afterward the plants were rehydrated. Leaves were taken from control plants after 7, 12, and 26 d from the beginning of dehydration, and after 6 and 48 h from rehydration. The RWC decreased by 80% during dehydration, but the leaves regained RWC with rehydration. Dehydrated plants showed lesser amounts of proteins, lipids, and chlorophyll, all of which increased following rewatering. The lipid-to-protein ratio, which decreased during dehydration, returned to control level after 48 h of rehydration. Thylakoid lipids were more unsaturated when RWC reached the value of 9%. EPR measurements of spin-labeled proteins showed the presence of three different groups of proteins with different mobility in thylakoid membranes. The rotational correlation time of groups 1 and 2 increased with dehydration and decreased upon rehydration, whereas group 3 showed little changes. Desiccation did not cause thylakoid swelling or breakage, but the membrane system assemblage showed changes in thylakoid stacking. After 48 h of rehydration the membrane system recovered completely the organization of the fully hydrated state, showing several well-defined and regularly distributed grana.     

The effects of cation-induced and pH-induced membrane stacking on chlorophyll fluorescence decay kinetics

We have compared the effects of thylakoid membrane appression by electrostatic screening and by charge neutralization on the room-temperature chlorophyll fluorescence decay kinetics of broken spinach chloroplasts. Monovalent and divalent metal cations induce both a structural differentiation of thylakoid membranes and a lateral segregation of pigment-protein complexes. These phenomena have distinct effects on the F0- and Fmax-level chlorophyll fluorescence decay kinetics at different levels of added cation. We further find specific cation effects, particularly on a 1-2 ns decay component at the Fmax fluorescence level, that are proposed to be related to the effectiveness of electrostatic screening as determined by the hydrated metal ionic radius. Distinct pH-induced effects on chlorophyll fluorescence decay kinetics are associated with the alternative mechanism of electrostatic neutralization to induce membrane stacking. These observations are used to construct a model of chlorophyll fluorescence emission that accounts for the variable kinetics and multiexponential character of the fluorescence decay upon membrane appression.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O₂) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O₂ reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H₂O₂) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H₂O₂ accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H₂O₂ from PETC has a potential of increasing mitochondrial respiration and CO₂ release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H₂O₂ accumulation. The putative production of H₂O₂ in any PETC location other than PSI is rapidly quenched and therefore cannot function in H₂O₂ translocation to another cellular location or in signaling.

Photosynthetically derived H₂O₂ only accumulates at Photosystem I and may trigger cooperation with mitochondria during stress.     

The degree of stacking and the average membrane area of thylakoids in lettuce chloroplasts

The surface density of stacked and total thylakoid membranes in chloroplasts was determined morphometrically using the method of vertical sections. The degree of stacking, defined as the fraction of the total membrane area which is involved in stacking, was calculated from the surface densities and found to be 0.70 for chloroplasts of lettuce grown under field conditions. An average membrane area of 500 microns 2 for the thylakoids in a chloroplast was obtained from the surface density and the average volume of the chloroplasts (11.6 microns 3, estimated by means of equivalent oblate spheroids). The advantage of the morphometric method over alternative techniques and the relevance of the results with respect to the topology of thylakoid membranes are discussed.     

A brief history of how microscopic studies led to the elucidation of the 3D architecture and macromolecular organization of higher plant thylakoids

Photosynthesis Research - Microscopic studies of chloroplasts can be traced back to the year 1678 when Antonie van Leeuwenhoek reported to the Royal Society in London that he saw green globules in...     

Phosphorylation of chloroplast thylakoids decreases the maximum capacity of photosystem-II electron transfer

Phosphorylation of chloroplast thylakoid polypeptides by the light-activated protein kinase was found to decrease the light-saturated rate of whole chain and Photosystem-II electron transport. This decrease in electron-transport capacity was reversible and was found to correlate with the phosphorylation-induced decrease in chlorophyll fluorescence.     

The stacking of chloroplast thylakoids: Evidence for segregation of charged groups into nonstacked regions

Small particles derived from the digitonin treatment of chloroplast thylakoid membranes in either the stacked (grana-containing) or unstacked condition, as determined by cation concentration, have been used to study the aggregation of thylakoid membranes. At pH values above 5, the small particles from stacked chloroplasts do not aggregate in the presence of Mg²⁺ or other screening cations at concentrations sufficient to cause the restacking of thylakoids from low-salt chloroplasts. However, the small particles from stacked chloroplasts are aggregated either by lowering the pH to 4.6 or adding the binding cation La³⁺. In contrast, the small particles obtained on digitonin treatment of unstacked chloroplasts were aggregated by cations at neutral pH. Large particles (mainly grana) derived from digitonin treatment of stacked chloroplasts could not be unstacked by transfer to media of low cation concentration. It is concluded that the nonappressed regions of the chloroplast thylakoid membranes under stacking conditions carry higher than average negative surface charge densities under physiological pH conditions. Transfer of chloroplasts to media of low cation concentration causes a time-dependent lateral redistribution of charge between the appressed and nonappressed regions, but this redistribution is prevented by prior digitonin treatment of stacked chloroplasts.     

Genetic analysis of chloroplast biogenesis in higher plants

The biogenesis of chloroplasts is genetically complex, involving hundreds of genes distributed between the nucleus and organelle. In higher plants, developmental parameters confer an added layer of complexity upon the genetic control of chloroplast biogenesis: the properties of plastids differ dramatically between different cell types. While the biochemistry and structure of different plastid types have been described in detail, factors that determine the timing and localization of chloroplast development and that mediate chloroplast assembly have remained elusive. To identify nuclear genes that play novel roles in chloroplast biogenesis, we are exploiting nuclear mutations that block the accumulation of subsets of chloroplast proteins. Detailed study of the mutant phenotypes provides clues concerning the primary defect in each mutant. Mutants with defects in chloroplast translation and mRNA metabolism have been identified. Other mutants defective in the accumulation of multiple thylakoid complexes show no apparent defect in the synthesis of the missing proteins. These may identify factors involved in the integration of proteins into the thylakoid membrane and their assembly into functional complexes.     

Microtubule patterns during meiosis in two higher plant species

Summary A monoclonal antibody to higher plant tubulin was used to trace microtubule (MT) structures by immunofluorescence throughout mitosis and meiosis in two angiosperms,Lycopersicon esculentum andOrnithogalum virens. Root tip cells showed stage specific MT patterns typical of higher plant cells. These included parallel cortical interphase arrays oriented perpendicular to the long axis of the cell, preprophase band MTs in late interphase through prophase, barrelshaped spindles, and finally phragmoplasts. Pollen mother cell divisions exhibited randomly oriented cortical MT arrays in prophase I, pointed spindles during karyokinesis, and elongate phragmoplasts. A preprophase band was not observed in either meiotic division. MT initiation sites were seen as broad zones associated with the nuclear envelope.     

Biochemical changes during sucrose deprivation in higher plant cells

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.     

The stacking of chloroplast thylakoids: Effects of cation screening and binding,studied by the digitonin method

The differential action of digitonin on stacked and unstacked chloroplast thylakoids was used to investigate the molecular interactions between thylakoid membranes. The yield of the heavy fraction which is obtained from chloroplasts after digitonin incubation and differential centrifugation was taken as a measure of the degree or tightness of membrane appression. The effects of various mono-, di-, and trivalent cations on the yield of the heavy fraction were studied, and the results interpreted in terms either of electrostatic screening or ion binding to the thylakoid membrane surface: Although there was some degree of cation specificity in the degree of thylakoid appression indicative of cation binding, the nonspecific screening effect was much more important in determining the overall balance of forces. It is postulated that stacking occurs in regions of low net surface charge density, with a possible segregation of excess negative charges into nonstacked regions.     

Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue.

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying the premature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs in appressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) as well as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacity for a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement of photochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimately intertwined.