Pea Chloroplasts Research Articles

Effect of the insecticide clothianidin on the photosynthetic electron transport chain in pea.

Clothianidin (CL) is a neonicotinoid insecticide widely used in crop protection against insect pests. However, its effects on photosynthesis remain largely unknown. Here, by investigating the influence of CL at the concentrations of 22 and 110 μg/L on the primary processes of photosynthesis, membrane fluidity and structural changes of pea chloroplasts, we located several primary binding sites of this pesticide. Similar dynamics were observed for both concentrations. However, statistically significant differences were only found at 110 μg/L for all methods used. The light saturated rate of linear electron flow decreased mainly due to the disturbance of electron flow on the acceptor side of photosystem II (PSII) associated with the appearance of QB-nonreducing centers and empty QB binding sites of PSII. The functioning of the donor side of PSII, the activity of photosystem I (PSI) and the maximum quantum yield of PSII photochemistry (Fv/Fm) were not found to be significantly altered. Increased membrane fluidity and structural alterations of the thylakoid membrane led to a decrease in the development of the proton gradient ΔрН and membrane energization processes.

PHYTOREMEDIATION OF PARTS CONTAMINATED WITH ISOPROTURONE, TRANSGENIC OILY BEANS

The widespread use of isoproturon [IPU] can lead to severe environmental pollution and threats to environmental functions. In this study, the PDMAB IPU bacterial n-demethylase gene was transported and expressed in fat pea chloroplast [Glycine Max L. 'Zhonghuang13']. Transgenic fat bean pods showed significant resistance to PPU and demethylated PPU to the phytotoxic 3-[4 - isopropylphenyl]-1-methylurea [MDIPU] metabolite in vivo. Transgenic fat bean pods removed 98% and 84% MPU from water and soil in 5 and 14 days respectively, accumulating fewer MPU in plant tissues compared to wild type [WT]. MPs showed higher rates of symbiotic nitrogen fixation in transgenic fat peas under stress [total biomass o f nodules and higher nitrogenase activity] and a more stable community of rhizosphere bacteria than WT. However, intensive use and accumulation of toxic herbicides can negatively affect the symbiosis of legumes. Thirty different herbicides and environmental pollutants have affected signaling between plants and rhizobias, delayed node formation, and reduced biological nitrogen fixation in vitro [Fox et al., 2007]. This study developed a transgenic [TS] oil bean that is capable of effectively removing PPU from the growing environment and restoring the high symbiotic ability to fix nitrogen under PPU stress, and provides new insight into the interaction between rhizospheric microorganisms and legumes under herbicidal stress.

Crucial role of the PTOX and CET pathways in optimizing ATP synthesis in mesophyll chloroplasts of C3 and C4 plants

Besides the linear electron flow (LET), several regulatory and alternative electron transfer pathways exist in chloroplasts, among them the cyclic electron transport around photosystem I (CET) and PTOX enzyme which oxidizes the plastoquinone pool. Until now has not been investigated the influence of PTOX activity on the regulation of CET pathways and hence the regulation of ATP production. The present study reveals differential responses of CET and PTOX regulation to environmental changes and how these pathways can adjust the ATP content to C3 and C4 metabolism. Here, we conducted a comparative study in maize (C4) and pea (C3) chloroplasts by measuring the activity of PSI in the presence of the inhibitors antimycin A and propyl gallate, estimating the content of thylakoid proteins that participate in CET, and determining ATP content and ATP synthase activity, and fluorescence analysis. We found that NDH-CET pathway plays an important role in maize chloroplasts, whereas PGR5/PGRL1 proteins play a dominant role in pea chloroplasts. The obtained results indicate a substantial involvement of PTOX activity in regulating electron flow in response to ATP demand. We also found that C3 and C4 chloroplasts contained quantitatively different CET membrane components. Based on the study findings, we propose a model of regulation of the linear electron transport (LET) and CET pathways in mesophyll chloroplasts of C4 and C3 plants under low light intensity based on ATP level and PTOX activity. • The NDH complex and PGR5/PGRL1 play a main role in CET in maize and pea mesophyll chloroplasts, respectively. • High PTOX activity in maize seems to be the key for producing ATP required for C4 metabolism. • PTOX is localized mainly in grana in maize and in stroma membranes in pea chloroplasts. • Antenna aggregation in maize indicates a high reduction, which may regulate ATP/NADPH demand.

Tic12, a 12-kDa essential component of the translocon at the inner envelope membrane of chloroplasts in Arabidopsis.

The complexes translocon at the outer envelope membrane of chloroplasts and translocon at the inner envelope membrane of chloroplasts (TIC) mediate preprotein translocation across the chloroplast outer and inner envelope membranes, respectively. Tic20, Tic56, Tic100, and Tic214 form a stable one-megadalton TIC whose function is essential for Arabidopsis thaliana and Chlamydomonas reinhardtii. Tic20 plays a central role in preprotein translocation by forming a protein-conducting channel. Tic56, Tic100, and Tic214 are also indispensable for TIC function, but whether other components are required for this process remain unclear. Here, we demonstrate that a 12-kDa protein named Tic12 is part of the TIC in A. thaliana and participates in preprotein translocation across the inner envelope membrane. Tic12 was tightly associated with the TIC but disassociated under high-salt conditions in combination with Triton X-100. Site-specific UV crosslinking experiments revealed that Tic12 and Tic20 directly interact with the transit peptide of a translocating preprotein. The tic12 null mutants are albino and seedling lethal, similar to the other tic null mutants. Tic12 and Tic20 were also involved in preprotein translocation in (Pisum sativum) pea chloroplasts. Thus, Tic12 is an essential constituent that forms the functional core together with Tic20 in the one-megadalton TIC.

Short-term heating causes thylakoid restructuring in pea chloroplasts and modifies spectral properties of pigment-protein complexes

Short-term heating causes thylakoid restructuring in pea chloroplasts and modifies spectral properties of pigment-protein complexes

The Effect of the Viscosity of a Trehalose Solution on ATP Hydrolysis by Chloroplast F1-ATPase

The effect of trehalose solution viscosity on the kinetics of ATP hydrolysis by CF1-ATPase (pea chloroplast F1-ATPase) was studied. Trehalose added to the reaction mixture had a double effect: the Mg-dependent enzyme activity was stimulated at trehalose levels below 20 wt % and suppressed at higher trehalose levels. The Ca-dependent activity of CF1 decreases monotonically with the increasing trehalose level. It was shown that stimulation of the Mg-dependent enzyme activity was a result of diminished Mg-ADP-dependent enzyme inactivation. In the case of negligible inactivation, the elevated trehalose content caused a decrease in the maximum rate of ATP hydrolysis and the apparent Michaelis constant increased. The changes in the values of the Michaelis constant and enzyme activity indicate that the delivery of reaction substrates and the conformational changes in CF1-ATPase accompanying the hydrolysis are impeded by the viscosity of the medium. In accordance with Cramer’s rule, the energy loss due to interaction with the medium leads to the conclusion that the efficiency of energy conversion by CF1-ATPase never reaches 100%.

Analysis of Changes in Photochemical Reflectance Index (PRI) in Relation to the Acidification of the Lumen of the Chloroplasts of Pea and Geranium Leaves under a Short-Term Illumination

Measurement of the photochemical reflectance index (PRI) is a simple and non-invasive method for the evaluation of photosynthetic processes in higher plants. At the same time, the relationship between photosynthetic parameters and PRI can be significantly modified at the initial stages of illumination; thus, the study of the mechanisms of development of PRI after the beginning of illumination is an important task. The aim of this work was to analyze the relationship between the acidification of chloroplast lumen, which was assessed as an increase in the absorption of light by the leaf at a wavelength of 535 nm (light scattering, LS), and changes in PRI under short-term illumination in the leaves of pea and geranium. It was shown that the illumination caused an increase in LS and a decrease in PRI in both studied objects. Significant differences were found for the amplitude of the photochemical reflectance index decrease, while the differences in the absolute values of PRI at different stages of illumination were not reliable. Correlation analysis showed that the increase in LS during the first two minutes of illumination, reflecting light-induced acidification of chloroplast lumen, strongly correlated with the amplitude of the decrease in PRI; at the same time, at later stages of illumination, such a relationship was absent. Additional analysis carried out on geranium showed that the cessation of illumination caused the opposite dynamics: the decrease in LS was accompanied by an increase in PRI. The results show that the changes in PRI at the first minutes after the beginning of illumination, apparently, were due to the acidification of the chloroplast lumen.

Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus.

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane β-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one β-barrel protein, Toc75, has been shown to use this pathway. The route other β-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that β-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

In Vitro Protein Import into Isolated Chloroplasts.

Chloroplasts contain about 3000 proteins, with approximately 400 of them located in the chloroplast envelope membranes, 1300 in the soluble stroma, and 1300 in the thylakoid membranes. Most of them are encoded by nuclear genes and translated as precursor proteins in the cytosol before their transport into chloroplasts. As a tool to control and characterize their import into plastids, we here describe an assay for in vitro protein import with isolated pea chloroplasts.

Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC-TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC-TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC-TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.

Glutathionylation of Pea Chloroplast 2-Cys Prx and Mitochondrial Prx IIF Affects Their Structure and Peroxidase Activity and Sulfiredoxin Deglutathionylates Only the 2-Cys Prx.

Together with thioredoxins (Trxs), plant peroxiredoxins (Prxs), and sulfiredoxins (Srxs) are involved in antioxidant defense and redox signaling, while their regulation by post-translational modifications (PTMs) is increasingly regarded as a key component for the transduction of the bioactivity of reactive oxygen and nitrogen species. Among these PTMs, S-glutathionylation is considered a protective mechanism against overoxidation, it also modulates protein activity and allows signaling. This study explores the glutathionylation of recombinant chloroplastic 2-Cys Prx and mitochondrial Prx IIF from Pisum sativum. Glutathionylation of the decameric form of 2-Cys Prx produced a change in the elution volume after FPLC chromatography and converted it to its dimeric glutathionylated form, while Prx IIF in its reduced dimeric form was glutathionylated without changing its oligomeric state. Mass spectrometry demonstrated that oxidized glutathione (GSSG) can glutathionylate resolving cysteine (Cys174), but not the peroxidatic equivalent (Cys52), in 2-Cys Prx. In contrast, GSSG was able to glutathionylate both peroxidatic (Cys59) and resolving (Cys84) cysteine in Prx IIF. Glutathionylation was seen to be dependent on the GSH/GSSG ratio, although the exact effect on the 2-Cys Prx and Prx IIF proteins differed. However, the glutathionylation provoked a similar decrease in the peroxidase activity of both peroxiredoxins. Despite growing evidence of the importance of post-translational modifications, little is known about the enzymatic systems that specifically regulate the reversal of this modification. In the present work, sulfiredoxin from P. sativum was seen to be able to deglutathionylate pea 2-Cys Prx but not pea Prx IIF. Redox changes during plant development and the response to stress influence glutathionylation/deglutathionylation processes, which may represent an important event through the modulation of peroxiredoxin and sulfiredoxin proteins.

Changes in H(+)-ATP Synthase Activity, Proton Electrochemical Gradient, and pH in Pea Chloroplast Can Be Connected with Variation Potential.

Local stimulation induces generation and propagation of electrical signals, including the variation potential (VP) and action potential, in plants. Burning-induced VP changes the physiological state of plants; specifically, it inactivates photosynthesis. However, the mechanisms that decrease photosynthesis are poorly understood. We investigated these mechanisms by measuring VP-connected systemic changes in CO2 assimilation, parameters of light reactions of photosynthesis, electrochromic pigment absorbance shifts, and light scattering. We reveal that inactivation of photosynthesis in the pea, including inactivation of dark and light reactions, was connected with the VP. Inactivation of dark reactions decreased the rate constant of the fast relaxation of the electrochromic pigment absorbance shift, which reflected a decrease in the H+-ATP synthase activity. This decrease likely contributed to the acidification of the chloroplast lumen, which developed after VP induction. However, VP-connected decrease of the proton motive force across the thylakoid membrane, possibly, reflected a decreased pH in the stroma. This decrease may be another mechanism of chloroplast lumen acidification. Overall, stroma acidification can decrease electron flow through photosystem I, and lumen acidification induces growth of fluorescence non-photochemical quenching and decreases electron flow through photosystem II, i.e., pH decreases in the stroma and lumen, possibly, contribute to the VP-induced inactivation of light reactions of photosynthesis.

Изменения состояния фотосинтетических мембран хлоропластов гороха при кратковременном прогреве

Изменения состояния фотосинтетических мембран хлоропластов гороха при кратковременном прогреве

Invitro reconstitution of co-translational D1 insertion reveals a role of the cpSec-Alb3 translocase and Vipp1in photosystem II biogenesis.

Photosystem II (PS II) is a multi-subunit complex localized in the thylakoid membrane that performs the light-dependent photosynthetic charge separation. The PS II reaction centre comprises, among others, the D1 protein. De novo synthesis and repair of PS II require efficient mechanisms for transport and insertion of plastid encoded D1 into the thylakoid membrane. To elucidate the process of D1 insertion, we used an invitro translation system derived from pea chloroplasts to reconstitute the D1 insertion. Thereby, truncated D1 encoding psbA mRNAs lacking a stop codon were translated in the presence of thylakoid membranes and the translation was stalled by addition of chloramphenicol. The generated ribosome nascent chain complexes (RNCs) were tightly associated with the thylakoids. Subsequently, these D1 insertion intermediates were enriched from solubilized thylakoids by sucrose cushion centrifugation. Immunological analyses demonstrated the presence of the cpSec translocase, Alb3, cpFtsY, cpSRP54 and Vipp1 (vesicle-inducing protein in plastids 1) in the enriched D1 insertion intermediates. A complex formation between cpSecY, Alb3, cpFtsY and Vipp1in thylakoid membranes was shown by gel filtration chromatography, BN (Blue Native)/SDS-PAGE and co-immunoprecipitation experiments. Furthermore, a stimulating effect of recombinant Vipp1 on the formation of a D1 insertion intermediate was observed invitro. These results suggest a co-operative function of these proteins in D1 insertion.

A new member of the psToc159 family contributes to distinct protein targeting pathways in pea chloroplasts.

Protein import into chloroplasts relies on specific targeting of preproteins from the cytosol to the organelles and coordinated translocation processes across the double envelope membrane. Here, two complex machineries constitute the so called general import pathway, which consists of the TOC and TIC complexes (translocon at the outer envelope of chloroplasts and translocon at the inner envelope of chloroplasts, respectively). The majority of canonical preproteins feature an N-terminal cleavable transit peptide, which is necessary for targeting and recognition at the chloroplast surface by receptors of TOC, where Toc159 acts as the primary contact site. We identified a non-canonical preprotein without the classical transit peptide, the superoxide dismutase (FSD1), which was then used in chemical crosslinking approaches to find new interaction partners at the outer envelope from pea chloroplasts. In this way we could link FSD1 to members of the Toc159 family in pea, namely psToc132 and psToc120. Using deletion mutants as well as a peptide scanning approach we defined regions of the preprotein, which are involved in receptor binding. These are distributed across the entire sequence; however the extreme N-terminus as well as a C-proximal domain turned out to be essential for targeting and import. En route into the plastid FSD1 engages components of the general import pathway, implying that in spite of the non-canonical targeting information and recognition by a specific receptor this preprotein follows a similar way across the envelope as the majority of plastid preproteins.

Diversity in Guanosine 3′,5′-Bisdiphosphate (ppGpp) Sensitivity among Guanylate Kinases of Bacteria and Plants

The guanosine 3',5'-bisdiphosphate (ppGpp) signaling system is shared by bacteria and plant chloroplasts, but its role in plants has remained unclear. Here we show that guanylate kinase (GK), a key enzyme in guanine nucleotide biosynthesis that catalyzes the conversion of GMP to GDP, is a target of regulation by ppGpp in chloroplasts of rice, pea, and Arabidopsis. Plants have two distinct types of GK that are localized to organelles (GKpm) or to the cytosol (GKc), with both enzymes being essential for growth and development. We found that the activity of rice GKpm in vitro was inhibited by ppGpp with a Ki of 2.8 μM relative to the substrate GMP, whereas the Km of this enzyme for GMP was 73 μM. The IC50 of ppGpp for GKpm was ∼10 μM. In contrast, the activity of rice GKc was insensitive to ppGpp, as was that of GK from bakers' yeast, which is also a cytosolic enzyme. These observations suggest that ppGpp plays a pivotal role in the regulation of GTP biosynthesis in chloroplasts through specific inhibition of GKpm activity, with the regulation of GTP biosynthesis in chloroplasts thus being independent of that in the cytosol. We also found that GKs of Escherichia coli and Synechococcus elongatus PCC 7942 are insensitive to ppGpp, in contrast to the ppGpp sensitivity of the Bacillus subtilis enzyme. Our biochemical characterization of GK enzymes has thus revealed a novel target of ppGpp in chloroplasts and has uncovered diversity among bacterial GKs with regard to regulation by ppGpp.

Plants Utilize a Highly Conserved System for Repair of NADH and NADPH Hydrates

NADH and NADPH undergo spontaneous and enzymatic reactions that produce R and S forms of NAD(P)H hydrates [NAD(P)HX], which are not electron donors and inhibit various dehydrogenases. In bacteria, yeast (Saccharomyces cerevisiae), and mammals, these hydrates are repaired by the tandem action of an ADP- or ATP-dependent dehydratase that converts (S)-NAD(P)HX to NAD(P)H and an epimerase that facilitates interconversion of the R and S forms. Plants have homologs of both enzymes, the epimerase homolog being fused to the vitamin B6 salvage enzyme pyridoxine 5'-phosphate oxidase. Recombinant maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) NAD(P)HX dehydratases (GRMZM5G840928, At5g19150) were able to reconvert (S)-NAD(P)HX to NAD(P)H in an ATP-dependent manner. Recombinant maize and Arabidopsis epimerases (GRMZM2G061988, At5g49970) rapidly interconverted (R)- and (S)-NAD(P)HX, as did a truncated form of the Arabidopsis epimerase lacking the pyridoxine 5'-phosphate oxidase domain. All plant NAD(P)HX dehydratase and epimerase sequences examined had predicted organellar targeting peptides with a potential second start codon whose use would eliminate the targeting peptide. In vitro transcription/translation assays confirmed that both start sites were used. Dual import assays with purified pea (Pisum sativum) chloroplasts and mitochondria, and subcellular localization of GFP fusion constructs in tobacco (Nicotiana tabacum) suspension cells, indicated mitochondrial, plastidial, and cytosolic localization of the Arabidopsis epimerase and dehydratase. Ablation of the Arabidopsis dehydratase gene raised seedling levels of all NADHX forms by 20- to 40-fold, and levels of one NADPHX form by 10- to 30-fold. We conclude that plants have a canonical two-enzyme NAD(P)HX repair system that is directed to three subcellular compartments via the use of alternative translation start sites.

Stereometrical analysis of number and size of prolamellar bodies during pea chloroplast development

The plastid prolamelar bodies in dark-grown pea seedlings undergo gradual transformation and decay after illumination with low intensity light. Random micrographs do not give direct information concerning the sizes and average numbers of prolamellar bodies in a plastid. These values were obtained after evaluation by a stereometrical method from the ratio of polamellar bodies sizes to the plastid size and from the frequency of prolamellar body sections of a given diameter. Plastids of dark-grown seedlings contained on the average at least one prolamellar body. After illumination the size of the bodies decreased rapidly owing to dispersion into primary thylakoids and split into much smaller numerous prolamellar bodies.

Multiple fates of non‐mature lumenal proteins in thylakoids

Most proteins found in the thylakoid lumen are synthesized in the cytosol with an N-terminal extension consisting of transient signals for chloroplast import and thylakoid transfer in tandem. The thylakoid-transfer signal is required for protein sorting from the stroma to thylakoids, mainly via the cpSEC or cpTAT pathway, and is removed by the thylakoidal processing peptidase in the lumen. An Arabidopsis mutant lacking one of the thylakoidal processing peptidase homologs, Plsp1, contains plastids with anomalous thylakoids and is seedling-lethal. Furthermore, the mutant plastids accumulate two cpSEC substrates (PsbO and PetE) and one cpTAT substrate (PsbP) as intermediate forms. These properties of plsp1-null plastids suggest that complete maturation of lumenal proteins is a critical step for proper thylakoid assembly. Here we tested the effects of inhibition of thylakoid-transfer signal removal on protein targeting and accumulation by examining the localization of non-mature lumenal proteins in the Arabidopsis plsp1-null mutant and performing a protein import assay using pea chloroplasts. In plsp1-null plastids, the two cpSEC substrates were shown to be tightly associated with the membrane, while non-mature PsbP was found in the stroma. The import assay revealed that inhibition of thylakoid-transfer signal removal did not disrupt cpSEC- and cpTAT-dependent translocation, but prevented release of proteins from the membrane. Interestingly, non-mature PetE2 was quickly degraded under light, and unprocessed PsbO1 and PsbP1 were found in a 440-kDa complex and as a monomer, respectively. These results indicate that the cpTAT pathway may be disrupted in the plsp1-null mutant, and that there are multiple mechanisms to control unprocessed lumenal proteins in thylakoids.

Mapping the signal peptide binding and oligomer contact sites of the core subunit of the pea twin arginine protein translocase.

Twin arginine translocation (Tat) systems of thylakoid and bacterial membranes transport folded proteins using the proton gradient as the sole energy source. Tat substrates have hydrophobic signal peptides with an essential twin arginine (RR) recognition motif. The multispanning cpTatC plays a central role in Tat operation: It binds the signal peptide, directs translocase assembly, and may facilitate translocation. An in vitro assay with pea (Pisum sativum) chloroplasts was developed to conduct mutagenesis and analysis of cpTatC functions. Ala scanning mutagenesis identified mutants defective in substrate binding and receptor complex assembly. Mutations in the N terminus (S1) and first stromal loop (S2) caused specific defects in signal peptide recognition. Cys matching between substrate and imported cpTatC confirmed that S1 and S2 directly and specifically bind the RR proximal region of the signal peptide. Mutations in four lumen-proximal regions of cpTatC were defective in receptor complex assembly. Copurification and Cys matching analyses suggest that several of the lumen proximal regions may be important for cpTatC-cpTatC interactions. Surprisingly, RR binding domains of adjacent cpTatCs directed strong cpTatC-cpTatC cross-linking. This suggests clustering of binding sites on the multivalent receptor complex and explains the ability of Tat to transport cross-linked multimers. Transport of substrate proteins cross-linked to the signal peptide binding site tentatively identified mutants impaired in the translocation step.