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1.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360890

RESUMO

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membranas Intracelulares/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Proteoma , Tilacoides/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Filogenia , Processamento de Proteína Pós-Traducional , Proteômica/métodos
2.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360743

RESUMO

Under natural environments, light quality and quantity are extremely varied. To respond and acclimate to such changes, plants have developed a multiplicity of molecular regulatory mechanisms. Non-photochemical quenching of chlorophyll fluorescence (NPQ) and thylakoid protein phosphorylation are two mechanisms that protect vascular plants. To clarify the role of thylakoid protein phosphorylation in energy-dependent quenching of chlorophyll fluorescence (qE) in rice plants, we used a direct Western blot assay after BN-PAGE to detect all phosphoproteins by P-Thr antibody as well as by P-Lhcb1 and P-Lhcb2 antibodies. Isolated thylakoids in either the dark- or the light-adapted state from wild type (WT) and PsbS-KO rice plants were used for this approach to detect light-dependent interactions between PsbS, PSII, and LHCII proteins. We observed that the bands corresponding to the phosphorylated Lhcb1 and Lhcb2 as well as the other phosphorylated proteins were enhanced in the PsbS-KO mutant after illumination. The qE relaxation became slower in WT plants after 10 min HL treatment, which correlated with Lhcb1 and Lhcb2 protein phosphorylation in the LHCII trimers under the same experimental conditions. Thus, we concluded that light-induced phosphorylation of PSII core and Lhcb1/Lhcb2 proteins is enhanced in rice PsbS-KO plants which might be due to more reactive-oxygen-species production in this mutant.


Assuntos
Clorofila/metabolismo , Fluorescência , Complexos de Proteínas Captadores de Luz/metabolismo , Luz , Oryza/metabolismo , Tilacoides/metabolismo , Clorofila/genética , Complexos de Proteínas Captadores de Luz/genética , Oryza/genética , Fosforilação , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/genética
3.
Biophys J ; 120(15): 3091-3102, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34214527

RESUMO

In green plants, light harvesting complex of Photosystem II (LHCII) absorbs and transports excitation energy toward the photosynthetic reaction centers and serves as a site for energy-dependent nonphotochemical quenching (qE), the photoprotective dissipation of energy as heat. LHCII is thought to activate dissipation through conformational changes that change the photophysical behaviors. Understanding this balance requires a characterization of how the conformations of LHCII, and thus its photophysics, are influenced by individual factors within the membrane environment. Here, we used ensemble and single-molecule fluorescence to characterize the excited-state lifetimes and switching kinetics of LHCII embedded in nanodisc- and liposome-based model membranes of various sizes and lipid compositions. As the membrane area decreased, the quenched population and the rate of conformational dynamics both increased because of interactions with other proteins, the aqueous solution, and/or disordered lipids. Although the conformational states and dynamics were similar in both thylakoid and asolectin lipids, photodegradation increased with thylakoid lipids, likely because of their charge and pressure properties. Collectively, these findings demonstrate the ability of membrane environments to tune the conformations and photophysics of LHCII.


Assuntos
Complexos de Proteínas Captadores de Luz , Tilacoides , Cinética , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II , Imagem Individual de Molécula , Tilacoides/metabolismo
4.
Nat Plants ; 7(8): 1119-1131, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239095

RESUMO

In green algae and plants, state transitions serve as a short-term light-acclimation process in the regulation of the light-harvesting capacity of photosystems I and II (PSI and PSII, respectively). During the process, a portion of light-harvesting complex II (LHCII) is phosphorylated, dissociated from PSII and binds with PSI to form the supercomplex PSI-LHCI-LHCII. Here, we report high-resolution structures of PSI-LHCI-LHCII from Chlamydomonas reinhardtii, revealing the mechanism of assembly between the PSI-LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM protein. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in supercomplex formation. The results unravel the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.


Assuntos
Adaptação Ocular/fisiologia , Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Fosforilação/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Isoformas de Proteínas/metabolismo , Clorofila/metabolismo , Estrutura Molecular , Tilacoides/metabolismo
5.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34063012

RESUMO

Single-walled carbon nanotubes (SWCNTs) emerge as promising novel carbon-based nanoparticles for use in biomedicine, pharmacology and precision agriculture. They were shown to penetrate cell walls and membranes and to physically interact and exchange electrons with photosynthetic complexes in vitro. Here, for the first time, we studied the concentration-dependent effect of foliar application of copolymer-grafted SWCNTs on the structural and functional characteristics of intact pea plants. The lowest used concentration of 10 mg L-1 did not cause any harmful effects on the studied leaf characteristics, while abundant epicuticular wax generation on both leaf surfaces was observed after 300 mg L-1 treatment. Swelling of both the granal and the stromal regions of thylakoid membranes was detected after application of 100 mg L-1 and was most pronounced after 300 mg L-1. Higher SWCNT doses lead to impaired photosynthesis in terms of lower proton motive force generation, slower generation of non-photochemical quenching and reduced zeaxanthin content; however, the photosystem II function was largely preserved. Our results clearly indicate that SWCNTs affect the photosynthetic apparatus in a concentration-dependent manner. Low doses (10 mg L-1) of SWCNTs appear to be a safe suitable object for future development of nanocarriers for substances that are beneficial for plant growth.


Assuntos
Cloroplastos/ultraestrutura , Nanotubos de Carbono/química , Ervilhas/fisiologia , Ervilhas/ultraestrutura , Fotossíntese , Folhas de Planta/anatomia & histologia , Dióxido de Carbono/metabolismo , Carotenoides/metabolismo , Permeabilidade da Membrana Celular , Clorofila/metabolismo , Fluorescência , Nanotubos de Carbono/ultraestrutura , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/ultraestrutura , Prótons , Tilacoides/metabolismo , Fatores de Tempo , Xantofilas/metabolismo
6.
Int J Mol Sci ; 22(10)2021 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-34067635

RESUMO

The color of bracts generally turns yellow or black from green during cereal grain development. However, the impact of these phenotypic changes on photosynthetic physiology during black bract formation remains unclear. Two oat cultivars (Avena sativa L.), 'Triple Crown' and 'Qinghai 444', with yellow and black bracts, respectively, were found to both have green bracts at the heading stage, but started to turn black at the flowering stage and become blackened at the milk stage for 'Qinghai 444'. Their photosynthetic characteristics were analyzed and compared, and the key genes, proteins and regulatory pathways affecting photosynthetic physiology were determined in 'Triple Crown' and 'Qinghai 444' bracts. The results show that the actual PSII photochemical efficiency and PSII electron transfer rate of 'Qinghai 444' bracts had no significant changes at the heading and milk stages but decreased significantly (p < 0.05) at the flowering stage compared with 'Triple Crown'. The chlorophyll content decreased, the LHCII involved in the assembly of supercomplexes in the thylakoid membrane was inhibited, and the expression of Lhcb1 and Lhcb5 was downregulated at the flowering stage. During this critical stage, the expression of Bh4 and C4H was upregulated, and the biosynthetic pathway of p-coumaric acid using tyrosine and phenylalanine as precursors was also enhanced. Moreover, the key upregulated genes (CHS, CHI and F3H) of anthocyanin biosynthesis might complement the impaired PSII activity until recovered at the milk stage. These findings provide a new insight into how photosynthesis alters during the process of oat bract color transition to black.


Assuntos
Avena/metabolismo , Flores/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Antocianinas/genética , Antocianinas/metabolismo , Clorofila/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/fisiologia , Tilacoides/metabolismo
7.
Nat Commun ; 12(1): 3475, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108457

RESUMO

How thylakoid membranes are generated to form a metabolically active membrane network and how thylakoid membranes orchestrate the insertion and localization of protein complexes for efficient electron flux remain elusive. Here, we develop a method to modulate thylakoid biogenesis in the rod-shaped cyanobacterium Synechococcus elongatus PCC 7942 by modulating light intensity during cell growth, and probe the spatial-temporal stepwise biogenesis process of thylakoid membranes in cells. Our results reveal that the plasma membrane and regularly arranged concentric thylakoid layers have no physical connections. The newly synthesized thylakoid membrane fragments emerge between the plasma membrane and pre-existing thylakoids. Photosystem I monomers appear in the thylakoid membranes earlier than other mature photosystem assemblies, followed by generation of Photosystem I trimers and Photosystem II complexes. Redistribution of photosynthetic complexes during thylakoid biogenesis ensures establishment of the spatial organization of the functional thylakoid network. This study provides insights into the dynamic biogenesis process and maturation of the functional photosynthetic machinery.


Assuntos
Membranas Intracelulares/metabolismo , Tilacoides/metabolismo , Proteínas de Bactérias/metabolismo , Membranas Intracelulares/ultraestrutura , Luz , Microscopia Eletrônica , Modelos Biológicos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Multimerização Proteica , Proteômica , Synechococcus/crescimento & desenvolvimento , Synechococcus/metabolismo , Synechococcus/ultraestrutura , Tilacoides/ultraestrutura
8.
Ecotoxicol Environ Saf ; 220: 112369, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34090109

RESUMO

Soil salinization seriously restricts the growth and yield of soybeans. However, little information is available on the early growth stages of soybeans which are subjected to the gibberellin biosynthesis inhibitor, prohexadione-calcium (Pro-Ca). This study aimed to investigate the effects of exogenous Pro-Ca on saline-alkali stress-induced damages to photosynthesis and antioxidant defenses in soybean (Glycine max L.) seedlings. At the V3 growth stage, salt-tolerant genotype Hefeng 50 (HF50) and salt-sensitive genotype Kenfeng 16 (KF16) were subjected to 110 mmol L-1 mixed saline-alkali stress respectively, and then 100 mg L-1 Pro-Ca was sprayed on the leaves. Our results showed that saline-alkali stress accelerated the degradation of thylakoids, inhibited chlorophyll synthesis, reduced shoot dry weight, electron transfer rate (ETR), and peroxidase (POD) activity, the concentration of ascorbic acid (AsA) and soluble sugar, but enhanced the concentration of proline, hydrogen peroxide (H2O2) and the rate of superoxide radical (O2∙-) generation. Additionally, saline-alkali stress induced a lower decrease of the net photosynthetic rate (Pn), potential activity of PSII (Fv/F0), and maximum quantum yield of PSII (Fv/Fm) in salt-tolerant HF50 than in salt-sensitive KF16. Nevertheless, foliar spraying of exogenous Pro-Ca increased the chlorophyll content, Pn, Fv/F0, and Fv/Fm. These results were more prominent when Pro-Ca was applied to KF16 under saline-alkali conditions. Furthermore, exogenous application of Pro-Ca retarded the degradation of thylakoids, increased the ETR and the accumulation of AsA, soluble sugar, and proline, activated the activities of superoxide dismutase (SOD), catalase (CAT), and POD, and decreased the concentration of malondialdehyde (MDA), electrolyte leakage (EL), O2∙-, and H2O2. These results indicated that Pro-Ca could effectively protect soybean seedlings against damage from saline-alkali stress by regulating seedling phenotype, photosynthetic apparatus, antioxidant defense, and osmoregulation.


Assuntos
Álcalis/toxicidade , Antioxidantes/metabolismo , Ácidos Cetoglutáricos/farmacologia , Fotossíntese/efeitos dos fármacos , Soja/efeitos dos fármacos , Clorofila/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Soja/crescimento & desenvolvimento , Soja/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Tilacoides/metabolismo
9.
J Plant Physiol ; 261: 153427, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33940557

RESUMO

Metamitron (MET) is a fruitlet thinning compound for apple trees, needing better understanding of its action on leaf energy metabolism, depending on nighttime temperature. A trial under environmental controlled conditions was set with 'Golden Reinders' potted trees, under 25/7.5 and 25/15 °C (diurnal/nighttime temperature), with (MET, 247.5 ppm) or without (CTR) application, and considering the monitoring of photosynthetic and respiration components from day 1 (D1) to 14 (D14). Net photosynthesis (Pn) decline promoted by MET after D1 was not stomatal related. Instead, non-stomatal constraints, reflected on the photosynthetic capacity (Amax), included a clear photosystem (PS) II inhibition (but barely of PSI), as shown by severe reductions in thylakoid electron transport at PSII level, maximal (Fv/Fm) and actual (Fv'/Fm') PSII photochemical efficiencies, estimate of quantum yield of linear electron transport (Y(II)), and the rise in PSII photoinhibition status (Fs/Fm' and PIChr) and uncontrolled energy dissipation (Y(NO)). To Pn inhibition also contributed the impact in RuBisCO along the entire experiment, regardless of night temperature, here reported for the first time. Globally, MET impact on the photosynthetic parameters was usually greater under 7.5 °C, with maximal impacts between D4 and D7, probably associated to a less active metabolism at lower temperature. Cellular energy metabolism was further impaired under 7.5 °C, through moderate inhibition of NADH-dependent malate dehydrogenase (MDH) and pyruvate kinase (PK) enzymes involved in respiration, in contrast with the increase of dark respiration in MET 7.5 until D7. The lower impact on PK and MDH under 15 °C and a likely global higher active metabolism at that temperature would agree with the lowest sucrose levels in MET 15 at D4 and D7. Our findings showed that MET alters the cell energy machinery in a temperature dependent manner, affecting the sucrose balance mainly at 15 °C, justifying the observed greater thinning potential.


Assuntos
Malus/metabolismo , Fotossíntese , Folhas de Planta/metabolismo , Temperatura , Triazinas/metabolismo , Dióxido de Carbono/metabolismo , Transporte de Elétrons/efeitos dos fármacos , Malus/efeitos dos fármacos , Fotoperíodo , Fotossíntese/efeitos dos fármacos , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/efeitos dos fármacos , Tilacoides/efeitos dos fármacos , Tilacoides/metabolismo , Triazinas/administração & dosagem
10.
J Exp Bot ; 72(13): 4938-4948, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33939808

RESUMO

During low light- (LL) induced state transitions in dark-adapted rice (Oryza sativa) leaves, light-harvesting complex (LHC) II become phosphorylated and associate with PSI complexes to form LHCII-PSI-LHCI supercomplexes. When the leaves are subsequently transferred to high light (HL) conditions, phosphorylated LHCII complexes are no longer phosphorylated. Under the HL-induced transition in LHC phosphorylation status, we observed a new green band in the stacking gel of native green-PAGE, which was determined to be LHCII aggregates by immunoblotting and 77K chlorophyll fluorescence analysis. Knockout mutants of protein phosphatase 1 (PPH1) which dephosphorylates LHCII failed to form these LHCII aggregates. In addition, the ability to develop non-photochemical quenching in the PPH1 mutant under HL was less than for wild-type plants. As determined by immunoblotting analysis, LHCII proteins present in LHCII-PSI-LHCI supercomplexes included the Lhcb1 and Lhcb2 proteins. In this study, we provide evidence suggesting that LHCII in the LHCII-PSI-LHCI supercomplexes are dephosphorylated and subsequently form aggregates to dissipate excess light energy under HL conditions. We propose that this LHCII aggregation, involving LHCII L-trimers, is a newly observed photoprotective light-quenching process operating in the early stage of acclimation to HL in rice plants.


Assuntos
Oryza , Clorofila , Complexos de Proteínas Captadores de Luz/metabolismo , Oryza/genética , Oryza/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Tilacoides/metabolismo
11.
Viruses ; 13(3)2021 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-33800072

RESUMO

Tomato spotted wilt virus (TSWV) is one of the most destructive plant viruses, causing severe losses in many important crops worldwide. The non-structural protein NSm of TSWV is a viral movement protein that induces viral symptoms. However, the molecular mechanisms by which NSm contributes to symptom development are unclear. Here, we present evidence that NSm directly interacts with Nicotiana benthamiana chloroplast thylakoid membrane protein TMP14 (NbTMP14) by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. The interaction between NSm and NbTMP14 led to the translocation of the NbTMP14 protein from the chloroplast to the cytoplasm in TSWV-infected plants, and overexpressing NSm decreased NbTMP14 mRNA accumulation. In addition, abnormal chloroplasts and starch accumulation were observed in TSWV-infected plants. Silencing of NbTMP14 by TRV VIGS also showed similar results to those of TSWV-infected plants. Overexpressing NbTMP14 in transgenic N. benthamiana plants impeded TSWV infection, and silencing NbTMP14 in N. benthamiana plants increased disease symptom severity and virus accumulation. To our knowledge, this is the first report showing that the plant chloroplast TMP14 protein is involved in viral infection. Knowledge of the interaction between NSm and NbTMP14 advances our understanding of the molecular mechanisms underlying TSWV symptom development and infection.


Assuntos
Cloroplastos/patologia , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas do Movimento Viral em Plantas/metabolismo , Tabaco/virologia , Tospovirus/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Sequência de Bases , Cloroplastos/virologia , Complexo de Proteína do Fotossistema I/genética , Doenças das Plantas/virologia , Proteínas do Movimento Viral em Plantas/genética , Análise de Sequência de DNA , Tilacoides/metabolismo , Proteínas não Estruturais Virais/genética
12.
J Agric Food Chem ; 69(32): 8910-8928, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-33793221

RESUMO

Being ubiquitously present in plants, microalgae, and cyanobacteria and as the major constituents of thylakoid membranes, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) make up approximately 52 and 26%, respectively, of chloroplast lipids. Thylakoid membranes harbor the photosynthetic complexes and numerous essential biochemical pathways where MGDG and DGDG play a central role in facilitating photosynthesis light reaction, maintaining chloroplast morphology, and responding to abiotic stresses. Furthermore, these galactolipids are also bioactive compounds with antitumor, antimicrobial, antiviral, immunosuppressive, and anti-inflammatory activities and important nutritional value. These characteristics are strictly dependent upon their fatty acyl chain length, olefinic nature, and stereoconfiguration. However, their application potentials are practically untapped, largely as a result of the fact that their availability in large quantity and high purity (structured galactolipids) is challenging. In addition to laborious extraction from natural sources, in vitro assembling of these molecules could be a promising alternative. Thus, this review updates the latest advances in elucidating biosynthesis paths of MGDG and DGDG and related enzyme systems, which present invaluable inspiration to design approaches for a retrosynthesis of galactolipids. More critically, this work summarizes recent developments in the biological and enzymatic syntheses of galactolipids, especially the strategic scenarios for the construction of in vitro enzymatic and/or chemoenzymatic synthesis routes. Protein engineering of enzymes involved in the synthesis of MGDG and DGDG to improve their properties is highlighted, and the applications of galactolipids in foods and medicine are also discussed.


Assuntos
Galactolipídeos , Complexo de Proteínas do Centro de Reação Fotossintética , Cloroplastos/metabolismo , Fotossíntese , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Plantas , Tilacoides/metabolismo
13.
Plant Cell ; 33(2): 248-269, 2021 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-33793824

RESUMO

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.


Assuntos
Membranas Intracelulares/metabolismo , Fotossíntese , Análise de Sequência de RNA , Synechocystis/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multiproteicos/metabolismo , Fotossíntese/genética , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Proteoma/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Tilacoides/metabolismo , Sistemas Toxina-Antitoxina , Transcriptoma/genética
14.
Biochem J ; 478(7): 1333-1346, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33687054

RESUMO

Photosystem I is the most efficient photosynthetic enzyme with structure and composition highly conserved among all oxygenic phototrophs. Cyanobacterial Photosystem I is typically associated into trimers for reasons that are still debated. Almost universally, Photosystem I contains a number of long-wavelength-absorbing 'red' chlorophylls (Chls), that have a sizeable effect on the excitation energy transfer and trapping. Here we present spectroscopic comparison of trimeric Photosystem I from Synechocystis PCC 6803 with a monomeric complex from the ΔpsaL mutant and a 'minimal' monomeric complex ΔFIJL, containing only subunits A, B, C, D, E, K and M. The quantum yield of photochemistry at room temperature was the same in all complexes, demonstrating the functional robustness of this photosystem. The monomeric complexes had a reduced far-red absorption and emission equivalent to the loss of 1.5-2 red Chls emitting at 710-715 nm, whereas the longest-wavelength emission at 722 nm was not affected. The picosecond fluorescence kinetics at 77 K showed spectrally and kinetically distinct red Chls in all complexes and equilibration times of up to 50 ps. We found that the red Chls are not irreversible traps at 77 K but can still transfer excitations to the reaction centre, especially in the trimeric complexes. Structure-based Förster energy transfer calculations support the assignment of the lowest-energy state to the Chl pair B37/B38 and the trimer-specific red Chl emission to Chls A32/B7 located at the monomer-monomer interface. These intermediate-energy red Chls facilitate energy migration from the lowest-energy states to the reaction centre.


Assuntos
Clorofila/metabolismo , Transferência de Energia , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Synechocystis/metabolismo , Tilacoides/metabolismo , Cinética , Multimerização Proteica
15.
Small ; 17(14): e2006608, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33690933

RESUMO

Natural photosynthetic "thylakoid" membranes found in green plants contain a large network of light-harvesting (LH) protein complexes. Rearrangement of this photosynthetic machinery, laterally within stacked membranes called "grana", alters protein-protein interactions leading to changes in the energy balance within the system. Preparation of an experimentally accessible model system that allows the detailed investigation of these complex interactions can be achieved by interfacing thylakoid membranes and synthetic lipids into a template comprised of polymerized lipids in a 2D microarray pattern on glass surfaces. This paper uses this system to interrogate the behavior of LH proteins at the micro- and nanoscale and assesses the efficacy of this model. A combination of fluorescence lifetime imaging and atomic force microscopy reveals the differences in photophysical state and lateral organization between native thylakoid and hybrid membranes, the mechanism of LH protein incorporation into the developing hybrid membranes, and the nanoscale structure of the system. The resulting model system within each corral is a high-quality supported lipid bilayer that incorporates laterally mobile LH proteins. Photosynthetic activity is assessed in the hybrid membranes versus proteoliposomes, revealing that commonly used photochemical assays to test the electron transfer activity of photosystem II may actually produce false-positive results.


Assuntos
Tilacoides , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral , Complexos de Proteínas Captadores de Luz/metabolismo , Lipídeos , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo
16.
Plant Cell Environ ; 44(6): 1816-1829, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33715163

RESUMO

Light is one of the most important environmental factors that affects various cellular processes in plant growth and development; it is also crucial for the metabolism of carbohydrates as it provides the energy source for photosynthesis. Under extended darkness conditions, carbon starvation responses are triggered by depletion of stored energy. Although light rapidly inhibits starvation responses, the molecular mechanisms by which light signalling affects this process remain largely unknown. In this study, we showed that the Arabidopsis thaliana light signalling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) are essential for plant survival after extended darkness treatment at both seedling and adult stages. Transmission electron microscopy analyses revealed that disruption of both FHY3 and FAR1 resulted in destruction of chloroplast envelopes and thylakoid membranes under extended darkness conditions. Furthermore, treatment with sucrose, but not glucose, completely rescued carbon starvation-induced cell death in the rosette leaves and arrested early seedling establishment in the fhy3 far1 plants. We thus concluded that the light signalling proteins FHY3 and FAR1 negatively regulate carbon starvation responses in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Carbono/metabolismo , Proteínas Nucleares/metabolismo , Fitocromo/metabolismo , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Morte Celular , Celulases/genética , Cloroplastos/metabolismo , Cloroplastos/patologia , Escuridão , Regulação da Expressão Gênica de Plantas , Glucose/metabolismo , Glucose/farmacologia , Mutação , Proteínas Nucleares/genética , Fitocromo/genética , Células Vegetais , Folhas de Planta/citologia , Plantas Geneticamente Modificadas , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sacarose/metabolismo , Sacarose/farmacologia , Tilacoides/metabolismo , Tilacoides/patologia
17.
Physiol Plant ; 172(4): 1983-1996, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33786842

RESUMO

Thylakoid membranes of young leaves consist of grana and stroma lamellae (stroma-grana [SG] structure). The SG thylakoid is gradually converted into isolated grana (IG), almost lacking the stroma lamellae during growth. This morphological alteration was found to cause a reduction in maximum photosynthetic rate and an enhancement of photoinhibition in photosystem II (PSII). In situ microspectrometric measurements of chlorophyll fluorescence in individual chloroplasts suggested an increase of the PSII/PSI ratio in IG thylakoids of mature leaves. Western blot analysis of isolated IG thylakoids showed relative increases in some PSII components, including the core protein (D1) and light-harvesting components CP24 and Lhcb2. Notably, a nonphotochemical quenching-related factor in the PSII supercomplex, PsbS, decreased by 40%. Changes in the high light response of PSII were detected through parameters of pulse-amplitude modulation fluorometry. Chlorophyll fluorescence lifetime indicated an increase of fluorescence quantum yield in IG. A minimal photodamage-repair rate analysis on a lincomycin treatment of the leaves indicated that repair rate constant of IG is slower than that of SG, while photodamage rate of IG is higher than that of SG. These results suggest that IG thylakoids are relatively sensitive to high light, which is not only due to a higher photodamage rate caused by some rearrangements of PS complexes, but also to the retarded PSII repair that may result from the lack of stroma lamellae. The IG thylakoids found among many plant species thus seem to be an adaptive form to low light environments, although their physiological roles still remain unclear.


Assuntos
Complexo de Proteína do Fotossistema II , Tilacoides , Clorofila/metabolismo , Cloroplastos/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Tilacoides/metabolismo
18.
Glob Chang Biol ; 27(13): 3133-3144, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33749034

RESUMO

In a rapidly warming world, we ask, "What limits the potential of marine diatoms to acclimate to elevated temperatures?," a group of ecologically successful unicellular eukaryotic photoautotrophs that evolved in a cooler ocean and are critical to marine food webs. To this end, we examined thermal tolerance mechanisms related to photosynthesis in the sequenced and transformable model diatom Phaeodactylum tricornutum. Data from transmission electron microscopy (TEM) and fatty acid methyl ester-gas chromatography mass spectrometry (FAME-GCMS) suggest that saturating thylakoid-associated fatty acids allowed rapid (on the order of hours) thermal tolerance up to 28.5°C. Beyond this critical temperature, thylakoid ultrastructure became severely perturbed. Biophysical analyses revealed that electrochemical leakage through the thylakoid membranes was extremely sensitive to elevated temperature (Q10 of 3.5). Data suggest that the loss of the proton motive force (pmf) occurred even when heat-labile photosystem II (PSII) was functioning, and saturation of thylakoid-associated fatty acids was active. Indeed, growth was inhibited when leakage of pmf through thylakoid membranes was insufficiently compensated by proton input from PSII. Our findings provide a mechanistic understanding of the importance of rapid saturation of thylakoid-associated fatty acids for ultrastructure maintenance and a generation of pmf at elevated temperatures. To the extent these experimental results apply, the ability of diatoms to generate a pmf may be a sensitive parameter for thermal sensitivity diagnosis in phytoplankton.


Assuntos
Diatomáceas , Tilacoides , Aclimatação , Ácidos Graxos/metabolismo , Fotossíntese , Força Próton-Motriz , Tilacoides/metabolismo
19.
Plant J ; 106(3): 766-784, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33583065

RESUMO

Copper (Cu) and iron (Fe) are essential for plant growth and are often in short supply under natural conditions. Molecular responses to simultaneous lack of both metals (-Cu-Fe) differ from those seen in the absence of either alone. Metabolome profiling of plant leaves previously revealed that fumarate levels fall under -Cu-Fe conditions. We employed lines lacking cytosolic FUMARASE2 (FUM2) activity to study the impact of constitutive suppression of cytosolic fumarate synthesis on plant growth under Cu and/or Fe deficiency. In fum2 mutants, photosynthesis and growth were less impaired under -Cu-Fe conditions than in wild-type (WT) seedlings. In particular, levels of photosynthetic proteins, chloroplast ultrastructure, amino acid profiles and redox state were less perturbed by simultaneous Cu-Fe deficiency in lines that cannot produce fumarate in the cytosol. Although cytosolic fumarate has been reported to promote acclimation of photosynthesis to low temperatures when metal supplies are adequate, the photosynthetic efficiency of fum2 lines grown under Cu-Fe deficiency in the cold was higher than in WT. Uptake and contents of Cu and Fe are similar in WT and fum2 plants under control and -Cu-Fe conditions, and lack of FUM2 does not alter the ability to sense metal deficiency, as indicated by marker gene expression. Collectively, we propose that reduced levels of cytosolic fumarate synthesis ultimately increase the availability of Fe for incorporation into metalloproteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cobre/deficiência , Ferro/deficiência , Fotossíntese , Aminoácidos/metabolismo , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/fisiologia , Cobre/metabolismo , Fumaratos/metabolismo , Ferro/metabolismo , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Tilacoides/metabolismo
20.
Nat Commun ; 12(1): 1100, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597543

RESUMO

Photosystem I (PSI) and II (PSII) balance their light energy distribution absorbed by their light-harvesting complexes (LHCs) through state transition to maintain the maximum photosynthetic performance and to avoid photodamage. In state 2, a part of LHCII moves to PSI, forming a PSI-LHCI-LHCII supercomplex. The green alga Chlamydomonas reinhardtii exhibits state transition to a far larger extent than higher plants. Here we report the cryo-electron microscopy structure of a PSI-LHCI-LHCII supercomplex in state 2 from C. reinhardtii at 3.42 Å resolution. The result reveals that the PSI-LHCI-LHCII of C. reinhardtii binds two LHCII trimers in addition to ten LHCI subunits. The PSI core subunits PsaO and PsaH, which were missed or not well-resolved in previous Cr-PSI-LHCI structures, are observed. The present results reveal the organization and assembly of PSI core subunits, LHCI and LHCII, pigment arrangement, and possible pathways of energy transfer from peripheral antennae to the PSI core.


Assuntos
Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Algas/química , Proteínas de Algas/ultraestrutura , Clorofila/metabolismo , Microscopia Crioeletrônica , Transferência de Energia , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/ultraestrutura , Modelos Moleculares , Fotossíntese , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/ultraestrutura , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Complexo de Proteína do Fotossistema II/ultraestrutura , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Tilacoides/metabolismo , Tilacoides/ultraestrutura
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