Vascular Plant Chloroplasts Research Articles

Structure, biogenesis, and evolution of thylakoid membranes.

Cyanobacteria and chloroplasts of algae and plants harbor specialized thylakoid membranes (TMs) that convert sunlight into chemical energy. These membranes house PSII and I, the vital protein-pigment complexes that drive oxygenic photosynthesis. In the course of their evolution, TMs have diversified in structure. However, the core machinery for photosynthetic electron transport remained largely unchanged, with adaptations occurring primarily in the light-harvesting antenna systems. Whereas TMs in cyanobacteria are relatively simple, they become more complex in algae and plants. The chloroplasts of vascular plants contain intricate networks of stacked grana and unstacked stroma thylakoids. This review provides an in-depth view of TM architectures in phototrophs and the determinants that shape their forms, as well as presenting recent insights into the spatial organization of their biogenesis and maintenance. Its overall goal is to define the underlying principles that have guided the evolution of these bioenergetic membranes.

László Mustárdy (1945-2022)

In photosynthesis research, in the early 1970s, one of the main problems was to understand the structure and functions of the thylakoid membranes of chloroplasts of vascular plants. Låszló Mustårdy, with his great skill and legendary patience, took care excellently of the tasks of electron microscopic ultrastructure.

RNA-stabilization factors in chloroplasts of vascular plants.

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants.

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens.

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.

The chloroplast ATP‐dependent Clp protease in vascular plants – new dimensions and future challenges

The ATP-dependent Clp protease is by far the most intricate protease in chloroplasts of vascular plants. Structurally, it is particularly complex with a proteolytic core complex containing 11 distinct subunits along with three potential chaperone partners. The Clp protease is also essential for chloroplast development and overall plant viability. Over the past decade, many of the important characteristics of this crucial protease have been revealed in the model plant species Arabidopsis thaliana. Despite this, challenges still remain in fully resolving certain key features, in particular, how the assembly of this multisubunit protease is regulated, the full range of native protein substrates and how they are targeted for degradation and how this complicated enzyme might have developed from simpler bacterial forms. This article focuses upon the recent advances in revealing the details underlying these important features. It also take the opportunity to speculate upon many of these findings in the hope of stimulating further investigation.

The Three-Dimensional Network of the Thylakoid Membranes in Plants: Quasihelical Model of the Granum-Stroma Assembly

The three-dimensional (3-D) network of the granum-stroma thylakoid assembly of vascular plant chloroplasts exhibits complex structural/functional heterogeneity. A complete understanding of the ultrastructure of this assembly is critical for our understanding of thylakoid function. The prevailing historical model of thylakoid structure, based on information derived from serial section analyses of electron microscopy (EM) images, suggests a helical arrangement of stroma membranes wound around the granum stacks. More recently, electron tomography has emerged as the leading method for the study of thylakoid ultrastructure, as it provides for higher resolution in the depth dimension. The first detailed 3D topological model derived from electron tomography was in disagreement with the helical model, whereas a more recent electron tomography study, conducted under somewhat different experimental conditions, suggested that basic features of the helical model are still valid. Here, we review the conventional EM data and present a critical discussion of the two electron tomography data sets in an attempt to establish a consensus model that accommodates all the information presently available.

Histochemical evidence for lipid A (endotoxin) in eukaryote chloroplasts.

Lipopolysaccharide (LPS) (a.k.a., endotoxin) is an essential component of the outer leaflet of the outer membrane of gram-negative bacteria and is a potent activator of the innate immune system of animals. Lipid A, the glycolipid core of LPS, is the agent responsible for disease and death from gram-negative sepsis, an important cause of human mortality and morbidity. Although it is generally accepted that lipid A is restricted to the prokaryotes, recent efforts to purify molecules from green algae with structural features unique to lipid A have met with success. Furthermore, the vascular plant Arabidopsis thaliana has been found to contain genes that encode all of the enzymes of the biosynthetic pathway for lipid A. It is not known whether vascular plants synthesize lipid A or where lipid A might be located in the tissues. For the present study, we used affinity reagents for lipid A to probe green alga and tissues of the garden pea for a light microscopic localization of lipid A in these eukaryote cells. We find staining for lipid A in free-living and endosymbiotic green algae and in the chloroplasts of vascular plants, indicating that this molecule is not restricted to prokaryotes, but is found also in select eukaryotes.

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes

Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.

Complex Formation of Vipp1 Depends on Its α-Helical PspA-like Domain

Vipp1 (vesicle-inducing protein in plastids 1) is found in Cyanobacteria and chloroplasts of photosynthetic eukaryotes where it is essential for the formation of the thylakoid membrane. Vipp1 is closely related to the phage shock protein A (PspA), a bacterial protein induced under diverse stress conditions. Vipp1 proteins differ from PspA by an additional C-terminal domain that is required for Vipp1 function in thylakoid biogenesis. We show here that in Cyanobacteria, green algae, and vascular plants, Vipp1 is part of a high molecular mass complex. The complex is formed by multiple copies of Vipp1, and complex formation involves interaction of the central alpha-helical domain that is common to Vipp1 as well as to PspA proteins. In chloroplasts of vascular plants, the Vipp1 complex can be visualized by green fluorescent protein fusion in discrete locations at the inner envelope. Green fluorescent protein fusion analysis furthermore revealed that complex formation is important for proper positioning of Vipp1 at the inner envelope of chloroplasts.

RNA binding-proteins interact specifically with the Arabidopsis chloroplast psbA mRNA 5' untranslated region in a redox-dependent manner.

The 5' untranslated region (5'UTR) of the psbA mRNA (psbA encodes the PSII reaction center protein, D1) is a key site for RNA-protein interactions in the post-transcriptional regulation of gene expression. In this study, we mapped the major psbA mRNA 5'-terminus at -77 nt, and two minor termini clusters centered at -48 and -64 nt, upstream from the psbA translational start codon of Arabidopsis thaliana. RNA mobility shift, RNase protection and UV-crosslinking assays were used to characterize the interaction of chloroplast proteins with the RNA 5'UTR. RNA-protein interactions depended upon a thermolabile secondary structure and specific sequences in a 35 nt region of the 5'UTR, which were 80% conserved with the psbA 5'UTRs from five other plants. Major and minor proteins of 43- and 30-kDa, respectively, were detected by UV-crosslinking to RNA. Oxidizing conditions abolished the association of the proteins with the 5'UTR, while RNA-binding activity was recovered upon incubation with a reductant. Based on these findings, we hypothesize that post-transcriptional regulation of psbA gene expression in chloroplasts of vascular plants involves redox-dependent interactions between specific sequences in the 5'UTR and 43- and 30-kDa RNA-binding proteins.

THE γ SUBUNITS OF PHYCOERYTHRIN FROM A RED ALGA: POSITION IN PHYCOBILISOMES AND SEQUENCE CHARACTERIZATION

Aglaothamnion neglectum Feldman‐Mazoyer has two γ subunits, γ31 and γ33, that are associated with phycoerythrin in the light‐harvesting phycobilisomes. We demonstrate that these subunits are spatially separated within the phycobilisome, with the γ31 subunit present at the distal end of phycobilisome rods and the γ33 subunit present on the proximal end. These subunits are thought to link phycoerythrin hexamers together in the rod substructure, serving a role analogous to that of linker polypeptides of cyanobacteria (although unlike the cyanobacterial linker polypeptides they are chromophorylated). The sequencing of tryptic polypeptides of the γ subunits enabled us to prepare oligonucleotides encoding different regions of γ31. These oligonucleotides were used as primers to generate a probe for isolating a γ31 cDNA clone. Characterization of the cDNA clone predicts a polypeptide of 280 amino acids with a 42 amino acid presequence that is characteristic of a transit peptide, the peptide that targets proteins to chloroplasts of vascular plants. The γ31 subunit has 50% similarity to the previously characterized γ33 subunit but has no identifiable similarity to functionally related polypeptides present in cyanobacterial phycobilisomes or to any other polypeptides in the databases. A repeat of 95 amino acids is present in the red algal γ subunit sequences, suggesting that these proteins were generated by a gene duplication followed by fusion of the duplicate sequences.

Patch-clamp study of vascular plant chloroplasts: ion channels and photocurrents.

This article presents direct measurements of large-conductance cation channel (G = 730 pS in 100/50 mM KCl; PK+/PCl- = 2.8) and light-induced current (photocurrent) in chloroplasts from C4 plants, Amaranthus hybridus and Zea mays, using the conventional patch-clamp technique. It was shown that preillumination of chloroplast gave rise to a fast decaying transient (tau approximately equal to 6 ms) in the light-induced current for the second and following light pulses. This transient increase of photocurrent was interpreted as a consequence of photoreducing of the redox pool between photosystems.

Spectroscopic methods in photosynthesis: photosystem stoichiometry and chlorophyll antenna size

Light-induced absorbance change and fluorescence measurements were employed in the quantitation of photosystem stoichiometry and in the measurement of the chlorophyll (Chl) antenna size in thylakoid membranes. Results with thylakoid membranes from diverse photosynthetic tissues indicated a PSII/PSI reaction-centre stoichiometry that deviates from unity. Cyanobacteria and red algae have a PSII/PSI ratio in the range of 0.3 to 0.7. Chloroplasts from spinach and other vascular-plant species grown under direct sunlight have PSII/PSI = 1.8±0.3. Chlorophyllb-deficient and Chib-lacking mutants have PSII/PSI > 2. The observation that PSII/PSI ratios are not unity and show a large variation among different photosynthetic membranes appears to be contrary to the conventional assumption derived from the Z-scheme. However, the photosystem stoichiometry is not the only factor that needs to be taken into account to explain the coordination of the two photosystems in the process of linear electron transport. The light-harvesting capacity of each photosystem must also be considered. In cyanobacterial thylakoids (fromSynechococcus6301, PSII/PSI = 0.5±0.2), the phycobilisome-PSII complexes collectively harvest as much light as the PSI complexes. In vascular plant chloroplasts, the light-harvesting capacity of a PSI I complex (250 molecules, Chi a/Chi b = 1.7) is lower than that of a PSI complex (230 Chl, Chla/Chlb= 8.0) because Chibhas a lower extinction coefficient than Chia. A differential attenuation of light intensity through the grana further reduces the light absorbed by PSII. Hence, a PSII/PSI ratio greater than one in vascular-plant chloroplasts compensates for the lower absorption of light by individual PSII complexes and ensures that, on average, PSII will harvest about as much light as PSI. In conclusion, distinct light-harvesting strategies among diverse plant species complement widely different photosystem stoichiometries to ensure a balanced absorption of light and a balanced electron flow between the two photoreactions, thereby satisfying the requirement set forth upon the formulation of the Z-scheme by Hill & Bendall (Nature, Lond.186, 136-137 (1960)) and by Duysens, Amesz & Kamp (Nature, Lond. 190, 510-511 (1961)).

Effect of inorganic phosphate, 3‐phosphoglycerate and glucose‐6‐phosphate on net starch synthesis by isolated chloroplasts of Codium fragile (Suringar) Hariot

Net starch synthesis isolated chloroplasts of Codium fragile appears to be regulated in a similar manner to that of vascular plants. Exogenous 10 mol m-3 PGA stimulated net starch synthesis at 200 μmol m-2 s -1 (PPFD) by approx. 69%, whilst 10 mol m-3 P1 not only inhibited synthesis but also promoted net starch degradation. However, unlike vascular plant chloroplasts, 10 mol m-3 G6P was found to stimulate net starch synthesis by approx. 52% under the same conditions. This apparent difference in response to exogenous G6P is discussed in relation to the operation of the P1 -G6P translocator in chloroplasts of this species.

Photosynthetic characteristics of Prochloron sp./ascidian symbioses

The prokaryotic green alga Prochloron sp. (Prochlorophyta) is found in symbiotic association with colonial didemnid ascidians that inhabit warm tropical waters in a broad range of light environments. We sought to determine the light-adaptation features of this alga in relation to the natural light environments in which the symbioses are found, and to characterize the temperature sensitivity of photosynthesis and respiration of Prochloron sp. in order to assess its physiological role in the productivity and distribution of the symbiosis. Colonies of the host ascidian Lissoclinum patella were collected from exposed and shaded habitats in a shallow lagoon in Palau, West Caroline Islands, during February and March, 1983. Some colonies from the two light habitats were maintained under conditions of high light (2 200 μE m−2 s−1) and low light (400 μE m−2 s−1) in running seawater tanks. The environments were characterized in terms of daily light quantum fluxes, daily periods of light-saturated photosynthesis (Hsat), and photon flux density levels. Prochloron sp. cells were isolated from the hosts and examined for their photosynthesis vs irradiance relationships, respiration, pigment content and photosynthetic unit features. In addition, daily P:R ratios, photosynthetic quotients, carbon balances and photosynthetic carbon release were also characterized. It was found that Prochloron sp. cells from low-light colonies possessed lower chlorophyll a/b ratios, larger photosynthetic units sizes based on both reaction I and reaction II, similar numbers of reaction center I and reaction center II per cell, lower respiration levels, and lower Pmax values than cells from high-light colonies. Cells isolated from low-light colonies showed photoinhibition of Pmax at photon flux densities above 800 μE m−2 s−1. However, because the host tissue attenuates about 60 to 80% of the incident irradiance, it is unlikely that these cells are normally photoinhibited in hospite. Collectively, the light-adaptation features of Prochloron sp. were more similar to those of eukaryotic algae and vascular plant chloroplasts than to those of cyanobacteria, and the responses were more sensitive to the daily flux of photosynthetic quantum than to photon flux density per se. Calculation of daily minimum carbon balances indicated that, though high-light cells had daily P:R ratios of 1.0 compared to 4.6 for low-light cells, the cells from the two different light environments showed nearly identical daily carbon gains. Cells isolated from high-light colonies released between 15 and 20% of their photosynthetically-fixed carbon, levels sufficient to be important in the nutrition of the host. Q10 responses of photosynthesis and respiration in Prochloron sp. cells exposed briefly (15–45 min) to temperatures between 15° and 45°C revealed a discontinuity in the photosynthetic response at the ambient growth temperatures. The photosynthetic rates were found to be more than twice as sensitive to temperatures below ambient (Q10=3.47) than to temperatures above ambient (Q10=1.47). The Q10 for respiration was constant (Q10=1.66) over the temperature range examined. It appears that the photosynthetic temperature sensitivity of Prochloron sp. may restrict its distribution to warmer tropical waters. The ecological implications of these findings are discussed in relation to published data on other symbiotic systems and free-living algae.