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1.
Biochim Biophys Acta Bioenerg ; 1861(1): 148086, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31678434

RESUMO

The Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), is coordinated by six carboxylate and one imidazole ligands. The roles of these ligands in the water oxidation mechanism remain largely unknown. In this study, we constructed a D1-D170H mutant, in which the Asp ligand bridging Mn and Ca ions was replaced with His, in the cyanobacterium Synechocystis sp. PCC 6803, and analyzed isolated PSII core complexes using Fourier transform infrared (FTIR) difference spectroscopy and mass spectrometry (MS). The S2-minus-S1 FTIR difference spectrum of the PSII complexes of the D1-D170H mutant showed features virtually identical to those of the wild-type PSII. MS analysis further showed that ~70% of D1 proteins from the PSII complexes of D1-D170H possessed the wild-type amino acid sequence, although only the mutated sequence was detected in genomic DNA in the same batch of cells for PSII preparations. In contrast, a D1-S169A mutant as a control showed a modified FTIR spectrum and only a mutated D1 protein. It is thus concluded that the FTIR spectrum of the D1-D170H mutant actually reflects that of wild-type PSII, whereas the Mn4CaO5 cluster is not formed in PSII with D1-D170H mutation. Although the mechanism of production of the wild-type D1 protein in the D1-D170H mutant is unknown at present, a caution is necessary in the analysis of site-directed mutants of crucial residues in the D1 protein, and mutation has to be confirmed not only at the DNA level but also at the amino acid level.


Assuntos
Proteínas de Bactérias/metabolismo , Mutação de Sentido Incorreto , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Espectrometria de Massas , Mutagênese Sítio-Dirigida , Complexo de Proteína do Fotossistema II/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Synechocystis/genética
2.
J Photochem Photobiol B ; 201: 111659, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31698219

RESUMO

Stressors of different natures, including drought stress, substantially compromise the ability of plants to effectively and safely utilize light energy. We investigated the influence of water stress on the photosynthetic processes in Picea abies and Pinus sylvestris, two species with contrasting drought sensitivities. Spruce and pine seedlings were exposed to polyethylene glycol 6000-induced water deficits of different intensities and durations. The maintenance of photosystem I (PSI) oxidation in spruce required increased photosynthetic control and led to the increased reduction of the plastoquinone pool, which was not the case in pine seedlings. As a result of increased excitation pressure, photosystem II (PSII) inactivation was observed in spruce plants, whereas in pine, the decreased PSII photochemistry was likely due to sustained non-photochemical quenching. Downregulation of PSII photochemistry and maintenance of PSI in an oxidized state were linked with the prevention of oxidative stress, even under severe water deficit. The decreased photosynthetic pigment content and photosynthetic gene expression suggested the coordinated downregulation of photosynthetic apparatus components under water stress to reduce light energy absorption. In summary, the observed adaptative mechanisms of pine and spruce to water stress may be similar to the well-studied adaptative mechanisms to winter stress, which may indicate the universality of protective mechanisms under various stresses in conifers.


Assuntos
Secas , Fotossíntese , Picea/metabolismo , Pinus sylvestris/metabolismo , Peroxidação de Lipídeos , Fotossíntese/genética , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Plântula/metabolismo
3.
Plant Sci ; 286: 1-6, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31300135

RESUMO

The excessive and harmful light energy absorbed by the photosystem (PS) II of higher plants is dissipated as heat through a protective mechanism termed non-photochemical quenching (NPQ) of chlorophyll fluorescence. PsbS-knock-out (KO) mutants lack the trans-thylakoid proton gradient (ΔpH)-dependent part of NPQ. To elucidate the molecular mechanism of NPQ, we investigated its dependency on oxygen. The development of NPQ in wild-type (WT) rice under low-oxygen (LO) conditions was reduced to more than 50% of its original value. However, under high-oxygen (HO) conditions, the NPQ of both WT and PsbS-KO mutants recovered. Moreover, WT and PsbS-KO mutant leaves infiltrated with the ΔpH dissipating uncoupler nigericin showed increased NPQ values under HO conditions. The experiments using intact chloroplasts and protoplasts of Arabidopsis thaliana supported that the LO effects observed in rice leaves were not due to carbon dioxide deficiency. There was a noticeable 90% reduction in the half-time of P700 oxidation rate in LO-treated leaves compared with that of WT control leaves, but the HO treatment did not significantly change the half-time of P700 oxidation rate. Overall, the results obtained here indicate that the stroma of the PsbS-KO plants could be potentially under O2 deficiency. Because the functions of PsbS in rice leaves are likely to be similar to those in other higher plants, our findings offer novel insights into the role of oxygen in the development of NPQ.


Assuntos
Adaptação Fisiológica/efeitos da radiação , Arabidopsis/metabolismo , Oryza/metabolismo , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/efeitos da radiação , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Oryza/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/genética , Folhas de Planta/fisiologia , Folhas de Planta/efeitos da radiação , Protoplastos/metabolismo , Protoplastos/efeitos da radiação
4.
J Photochem Photobiol B ; 197: 111535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31319267

RESUMO

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 µmol m-2 s-1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73-81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.


Assuntos
Proteínas de Arabidopsis/metabolismo , Clorofila A/química , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/fisiologia , Luz , Modelos Teóricos , Mutagênese , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/química , Teoria Quântica , Termodinâmica
5.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340536

RESUMO

Molecular mechanisms that are the base of the strategies adopted by Mediterranean plants to cope with the challenges imposed by limited or excessive solar radiation during the summer season have received limited attention. In our study, conducted on C. incanus plants growing in the shade or in full sunlight, we performed measurements of relevant physiological traits, such as leaf water potential, gas exchange and PSII photochemistry, RNA-Seq with de-novo assembly, and the analysis of differentially expressed genes. We also identified and quantified photosynthetic pigments, abscisic acid, and flavonoids. Here, we show major mechanisms regulating light perception and signaling which, in turn, sustain the shade avoidance syndrome displayed by the 'sun loving' C. incanus. We offer clear evidence of the detrimental effects of excessive light on both the assembly and the stability of PSII, and the activation of a suite of both repair and effective antioxidant mechanisms in sun-adapted leaves. For instance, our study supports the view of major antioxidant functions of zeaxanthin in sunny plants concomitantly challenged by severe drought stress. Finally, our study confirms the multiple functions served by flavonoids, both flavonols and flavanols, in the adaptive mechanisms of plants to the environmental pressures associated to Mediterranean climate.


Assuntos
Adaptação Biológica/efeitos dos fármacos , Cistus/efeitos da radiação , Regulação da Expressão Gênica de Plantas , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/efeitos da radiação , RNA de Plantas/genética , Ácido Abscísico/metabolismo , Adaptação Biológica/genética , Antioxidantes/metabolismo , Clorofila/biossíntese , Cistus/genética , Cistus/metabolismo , Dano ao DNA , Reparo do DNA , DNA de Plantas/genética , DNA de Plantas/metabolismo , Flavonoides/biossíntese , Transdução de Sinal Luminoso/genética , Região do Mediterrâneo , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , RNA de Plantas/metabolismo , Análise de Sequência de RNA , Energia Solar , Luz Solar , Água/metabolismo , Zeaxantinas/biossíntese
6.
Planta ; 250(2): 589-601, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31134341

RESUMO

MAIN CONCLUSION: The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.


Assuntos
Genoma de Cloroplastos/genética , Genoma de Planta/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Tabaco/genética , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Fosforilação , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteômica , Treonina/metabolismo , Tabaco/fisiologia
7.
Int J Mol Sci ; 20(5)2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836615

RESUMO

Early leaf senescence is an important agronomic trait that affects crop yield and quality. To understand the molecular mechanism of early leaf senescence, Oryza sativa premature leaf senescence 1 (ospls1) mutant rice with a deletion of OsVHA-A and its wild type were employed in this study. The genotype-dependent differences in photosynthetic indexes, senescence-related physiological parameters, and yield characters were investigated during the grain-filling stage. Moreover, RNA sequencing (RNA-seq) was performed to determine the genotype differences in transcriptome during the grain-filling stage. Results showed that the ospls1 mutant underwent significant decreases in the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), net photosynthesis rate (Pn), and soluble sugar and protein, followed by the decreases in OsVHA-A transcript and vacuolar H⁺-ATPase activity. Finally, yield traits were severely suppressed in the ospls1 mutant. RNA-seq results showed that 4827 differentially expressed genes (DEGs) were identified in ospls1 mutant between 0 day and 14 days, and the pathways of biosynthesis of secondary metabolites, carbon fixation in photosynthetic organisms, and photosynthesis were downregulated in the senescing leaves of ospls1 mutant during the grain-filling stage. In addition, 81 differentially expressed TFs were identified to be involved in leaf senescence. Eleven DEGs related to hormone signaling pathways were significantly enriched in auxin, cytokinins, brassinosteroids, and abscisic acid pathways, indicating that hormone signaling pathways participated in leaf senescence. Some antioxidative and carbohydrate metabolism-related genes were detected to be differentially expressed in the senescing leaves of ospls1 mutant, suggesting that these genes probably play response and regulatory roles in leaf senescence.


Assuntos
Senescência Celular/genética , Oryza/genética , Folhas de Planta/genética , Transcriptoma/genética , Clorofila/genética , Grão Comestível/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , Genótipo , Oryza/crescimento & desenvolvimento , Fenótipo , Fotossíntese/genética , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/crescimento & desenvolvimento , ATPases Translocadoras de Prótons/genética , Análise de Sequência de RNA , Transdução de Sinais , ATPases Vacuolares Próton-Translocadoras/genética
8.
Physiol Plant ; 166(1): 105-119, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30834537

RESUMO

The maximum quantum yield of photosystem II (as reflected by variable to maximum chlorophyll a fluorescence, Fv /Fm ) is regarded as one of the most important photosynthetic parameters. The genetic basis underlying natural variation in Fv /Fm , which shows low level of variations in plants under non-stress conditions, is not easy to be exploited using the conventional gene cloning approaches. Thus, in order to answer this question, we have followed another strategy: we used genome-wide association study (GWAS) and transgenic analysis in a rice mini-core collection. We report here that four single-nucleotide polymorphisms, located in the promoter region of ß-glucosidase 5 (BGlu-5), are associated with observed variation in Fv /Fm . Indeed, our transgenic analysis showed a good correlation between BGlu-5 and Fv /Fm . Thus, our work demonstrates the feasibility of using GWAS to study natural variation in Fv /Fm , suggesting that cis-element polymorphism, affecting the BGlu-5 expression level, may, indirectly, contribute to Fv /Fm variation in rice through the gibberellin signaling pathway. Further research is needed to understand the mechanism of our novel observation.


Assuntos
Estudo de Associação Genômica Ampla/métodos , Glucosidases/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Celulases/genética , Celulases/metabolismo , Giberelinas/metabolismo , Glucosidases/genética , Complexo de Proteína do Fotossistema II/genética , Polimorfismo de Nucleotídeo Único/genética
9.
Biochemistry ; 58(10): 1379-1387, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30707571

RESUMO

In photosystem II (PSII), photosynthetic water oxidation occurs at the tetramanganese-calcium cluster that cycles through light-induced intermediates (S0-S4) to produce oxygen from two substrate waters. The surrounding hydrogen-bonded amino acid residues and waters form channels that facilitate proton transfer and substrate water delivery, thereby ensuring efficient water oxidation. The residue D1-S169 lies in the "narrow" channel and forms hydrogen bonds with the Mn4CaO5 cluster via waters W1 and Wx. To probe the role of the narrow channel in substrate-water binding, we studied the D1-S169A mutation. PSII core complexes isolated from mutant cells exhibit inefficient S-state cycling and delayed oxygen evolution. The S2-state multiline EPR spectrum of D1-S169A PSII core complexes differed significantly from that of wild-type, and FTIR difference spectra showed that the mutation strongly perturbs the extensive network of hydrogen bonds that extends at least from D1-Y161 (YZ) to D1-D61. These results imply a possible role of D1-S169 in proton egress or substrate water delivery.


Assuntos
Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Aminoácidos/metabolismo , Cálcio/metabolismo , Ligações de Hidrogênio , Manganês/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema II/fisiologia , Prótons , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Água/química
10.
Biochim Biophys Acta Bioenerg ; 1860(4): 297-309, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30703365

RESUMO

The monomeric chlorophyll, ChlD1, which is located between the PD1PD2 chlorophyll pair and the pheophytin, PheoD1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg2+ is liganded to a water molecule that is H-bonded to D1/T179. Here, two site-directed mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn4CaO5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *ChlD1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of PD1PD2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the ChlD1+PheoD1- radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of 1O2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to ChlD1 upon QA- formation, indicates that in the T179H-PSII and in the WT*3-PSII, the ChlD1 itself is the chlorophyll that is first damaged by 1O2, whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, ChlD1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of ChlD1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.


Assuntos
Proteínas de Bactérias/química , Clorofila/química , Cianobactérias/enzimologia , Luz , Complexo de Proteína do Fotossistema II/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clorofila/genética , Clorofila/metabolismo , Cianobactérias/genética , Transporte de Elétrons/fisiologia , Mutagênese Sítio-Dirigida , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo
11.
Proc Natl Acad Sci U S A ; 116(9): 3385-3390, 2019 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-30808735

RESUMO

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.


Assuntos
Transferência de Energia/genética , Microalgas/metabolismo , Fotossíntese/genética , Zeaxantinas/química , Carotenoides/genética , Carotenoides/metabolismo , Clorofila/química , Clorofila/genética , Clorofila/metabolismo , Luz , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Microalgas/química , Microalgas/genética , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Xantofilas/química , Xantofilas/genética , Xantofilas/metabolismo , Zeaxantinas/genética , Zeaxantinas/metabolismo
12.
J Photochem Photobiol B ; 193: 18-30, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30798151

RESUMO

Carotenoids as part of the photosystems are crucial for their assembly, light-harvesting, and photoprotection. Light of different wavelengths impacts the composition and structure of photosystems, thus offering the possibility to influence the carotenoid concentrations and composition in photosystems by illumination with specific narrow-banded light spectra. Key components involved in the regulation of gene transcription are still poorly characterized, particularly in leafy vegetables as compared to model plants. In particular, the effect of different light qualities and its connection to redox control mechanisms, which also determine the photosystem composition and structure, is not yet well understood. Furthermore, light quality effects are species-dependent, and thus, increase the need to perform research on individual vegetable species such as pak choi Brassica rapa ssp. chinensis. Here, we investigated the carotenoid concentrations and composition of pak choi sprouts grown for 6 days under blue, red, or white light emitting diodes (LEDs) as light source. After 6 days, the total carotenoid content was the highest under white and slightly reduced under blue or red LEDs. Blue, red, and white light differently affected the carotenoid composition mainly due to variations of the ß-carotene content which could be correlated to changes in the transcript levels of ß-carotene hydroxylase 1 (ß-OHASE1). Further investigations implied a redox controlled gene expression of ß-OHASE1. In addition, transcription factors related to light signaling and the circadian clock differed in their transcriptional abundance after exposure to blue and red light. RNA-Seq analysis also revealed increased transcript levels of genes encoding the outer antenna complex of photosystem II under red compared to blue light, indicating an adjustment of the photosystems to the different light qualities which possibly contributed to the alternations in the carotenoid content and composition.


Assuntos
Brassica rapa/efeitos da radiação , Carotenoides/biossíntese , Luz , Sítios de Ligação , Brassica rapa/química , Brassica rapa/crescimento & desenvolvimento , Carotenoides/análise , Cromatografia Líquida de Alta Pressão , Relógios Circadianos/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Espectrometria de Massas , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
13.
Photosynth Res ; 140(1): 77-92, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30607859

RESUMO

In diverse terrestrial cyanobacteria, Far-Red Light Photoacclimation (FaRLiP) promotes extensive remodeling of the photosynthetic apparatus, including photosystems (PS)I and PSII and the cores of phycobilisomes, and is accompanied by the concomitant biosynthesis of chlorophyll (Chl) d and Chl f. Chl f synthase, encoded by chlF, is a highly divergent paralog of psbA; heterologous expression of chlF from Chlorogloeopsis fritscii PCC 9212 led to the light-dependent production of Chl f in Synechococcus sp. PCC 7002 (Ho et al., Science 353, aaf9178 (2016)). In the studies reported here, expression of the chlF gene from Fischerella thermalis PCC 7521 in the heterologous system led to enhanced synthesis of Chl f. N-terminally [His]10-tagged ChlF7521 was purified and identified by immunoblotting and tryptic-peptide mass fingerprinting. As predicted from its sequence similarity to PsbA, ChlF bound Chl a and pheophytin a at a ratio of ~ 3-4:1, bound ß-carotene and zeaxanthin, and was inhibited in vivo by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Cross-linking studies and the absence of copurifying proteins indicated that ChlF forms homodimers. Flash photolysis of ChlF produced a Chl a triplet that decayed with a lifetime (1/e) of ~ 817 µs and that could be attributed to intersystem crossing by EPR spectroscopy at 90 K. When the chlF7521 gene was expressed in a strain in which the psbD1 and psbD2 genes had been deleted, significantly more Chl f was produced, and Chl f levels could be further enhanced by specific growth-light conditions. Chl f synthesized in Synechococcus sp. PCC 7002 was inserted into trimeric PSI complexes.


Assuntos
Carbono-Oxigênio Ligases/metabolismo , Clorofila/análogos & derivados , Cianobactérias/enzimologia , Complexo de Proteína do Fotossistema I/metabolismo , Synechococcus/enzimologia , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/isolamento & purificação , Clorofila/metabolismo , Clorofila A/metabolismo , Cianobactérias/genética , Cianobactérias/fisiologia , Cianobactérias/efeitos da radiação , Expressão Gênica , Variação Genética , Luz , Mutagênese Sítio-Dirigida , Feofitinas/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Ficobilissomas , Synechococcus/genética , Synechococcus/fisiologia , Synechococcus/efeitos da radiação
14.
J Agric Food Chem ; 67(5): 1353-1359, 2019 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-30640451

RESUMO

There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to metribuzin in a wild radish population. Dose-response studies revealed a higher level of resistance to metribuzin in the resistant (R) compared to the susceptible (S) population. Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In addition, a higher level of [14C]-metribuzin metabolism and, consequently, a lower level of [14C] translocation were also detected in the R plants. These results demonstrated that both psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this wild radish population. Furthermore, this resistant population showed resistance to ALS-inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.


Assuntos
Resistência a Herbicidas , Herbicidas/farmacologia , Complexo de Proteína do Fotossistema II/genética , Proteínas de Plantas/genética , Raphanus/genética , Raphanus/metabolismo , Triazinas/farmacologia , Mutação , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Raphanus/efeitos dos fármacos
15.
Physiol Plant ; 165(4): 711-727, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29774565

RESUMO

Microdochium nivale is a fungal pathogen that causes yield losses of cereals during winter. Cold hardening under light conditions induces genotype-dependent resistance of a plant to infection. We aim to show how photosystem II (PSII) regulation contributes to plant resistance. Using mapping population of triticale doubled haploid lines, three M. nivale strains and different infection assays, we demonstrate that plants that maintain a higher maximum quantum efficiency of PSII show less leaf damage upon infection. The fungus can establish necrotrophic or biotrophic interactions with susceptible or resistant genotypes, respectively. It is suggested that local inhibition of photosynthesis during the infection of sensitive genotypes is not balanced by a supply of energy from the tissue surrounding the infected cells as efficiently as in resistant genotypes. Thus, defence is limited, which in turn results in extensive necrotic damage. Quantitative trait loci regions, involved in the control of both PSII functioning and resistance, were located on chromosomes 4 and 6, similar to a wide range of PSII- and resistance-related genes. A meta-analysis of microarray experiments showed that the expression of genes involved in the repair and de novo assembly of PSII was maintained at a stable level. However, to establish a favourable energy balance for defence, genes encoding PSII proteins resistant to oxidative degradation were downregulated to compensate for the upregulation of defence-related pathways. Finally, we demonstrate that the structural and functional integrity of the plant is a factor required to meet the energy demand of infected cells, photosynthesis-dependent systemic signalling and defence responses.


Assuntos
Ascomicetos/patogenicidade , Complexo de Proteína do Fotossistema II/metabolismo , Triticale/metabolismo , Triticale/microbiologia , Regulação da Expressão Gênica de Plantas , Fotossíntese/genética , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema II/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Locos de Características Quantitativas/genética , Triticale/genética
16.
Photosynth Res ; 139(1-3): 475-486, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29779191

RESUMO

Cytb559 in Photosystem II is a heterodimeric b-type cytochrome. The subunits, PsbE and PsbF, consist each in a membrane α-helix. Mutants were previously designed and studied in Thermosynechococcus elongatus (Sugiura et al., Biochim Biophys Acta 1847:276-285, 2015) either in which an axial histidine ligand of the haem-iron was substituted for a methionine, the PsbE/H23M mutant in which the haem was lacking, or in which the haem environment was modified, the PsbE/Y19F and PsbE/T26P mutants. All these mutants remained active showing that the haem has no structural role provided that PsbE and PsbF subunits are present. Here, we have carried on the characterization of these mutants. The following results were obtained: (i) the Y19F mutation hardly affect the Em of Cytb559, whereas the T26P mutation converts the haem into a form with a Em much below 0 mV (so low that it is likely not reducible by QB-) even in an active enzyme; (ii) in the PsbE/H23M mutant, and to a less extent in PsbE/T26P mutant, the electron transfer efficiency from QA- to QB is decreased; (iii) the lower Em of the QA/QA- couple in the PsbE/H23M mutant correlates with a higher production of singlet oxygen; (iv) the superoxide and/or hydroperoxide formation was not increased in the PsbE/H23M mutant lacking the haem, whereas it was significantly larger in the PsbE/T26P. These data are discussed in view of the literature to discriminate between structural and redox roles for the haem of Cytb559 in the production of reactive oxygen species.


Assuntos
Cianobactérias/metabolismo , Grupo dos Citocromos b/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Cianobactérias/genética , Grupo dos Citocromos b/química , Grupo dos Citocromos b/genética , Mutação/genética , Oxirredução , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Espécies Reativas de Oxigênio/metabolismo
17.
Pest Manag Sci ; 75(1): 144-151, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29797480

RESUMO

BACKGROUND: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides. RESULTS: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides. CONCLUSION: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides. © 2018 Society of Chemical Industry.


Assuntos
Atrazina/farmacologia , Diurona/farmacologia , Resistência a Herbicidas/genética , Herbicidas/farmacologia , Complexo de Proteína do Fotossistema II/genética , Raphanus/genética , Triazinas/farmacologia , Mutação , Complexo de Proteína do Fotossistema II/metabolismo , Raphanus/efeitos dos fármacos , Austrália Ocidental
18.
Methods ; 155: 30-40, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30503825

RESUMO

Transcripts have intrinsic propensity to form stable secondary structure that is fundamental to regulate RNA transcription, splicing, translation, RNA localization and turnover. Numerous methods that integrate chemical reactions with next-generation sequencing (NGS) have been applied to study in vivo RNA structure, providing new insights into RNA biology. Dimethyl sulfate (DMS) probing coupled with mutational profiling through NGS (DMS-MaPseq) is a newly developed method for revealing genome-wide or target-specific RNA structure. Herein, we present our experimental protocol of a modified DMS-MaPseq method for plant materials. The DMS treatment condition was optimized, and library preparation procedures were simplified. We also provided custom scripts for bioinformatic analysis of genome-wide DMS-MaPseq data. Bioinformatic results showed that our method could generate high-quality and reproducible data. Further, we assessed sequencing depth and coverage for genome-wide RNA structure profiling in Arabidopsis, and provided two examples of in vivo structure of mobile RNAs. We hope that our modified DMS-MaPseq method will serve as a powerful tool for analyzing in vivo RNA structurome in plants.


Assuntos
Arabidopsis/genética , Biologia Computacional/métodos , Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/química , RNA de Plantas/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pareamento de Bases , Sequência de Bases , Complexos de Proteínas Captadores de Luz/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Mutação , Conformação de Ácido Nucleico , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Ésteres do Ácido Sulfúrico/química
19.
Mol Plant ; 12(1): 86-98, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30453087

RESUMO

Photosystem II (PSII) core phosphatase (PBCP) selectively dephosphorylates PSII core proteins including D1, D2, CP43, and PsbH. PBCP function is required for efficient degradation of the D1 protein in the repair cycle of PSII, a supramolecular machinery highly susceptible to photodamage during oxygenic photosynthesis. Here we present structural and functional studies of PBCP from Oryza sativa (OsPBCP). In a symmetrical homodimer of OsPBCP, each monomer contains a PP2C-type phosphatase core domain, a large motif characteristic of PBCPs, and two small motifs around the active site. The large motif contributes to the formation of a substrate-binding surface groove, and is crucial for the selectivity of PBCP toward PSII core proteins and against the light-harvesting proteins. Remarkably, the phosphatase activity of OsPBCP is strongly inhibited by glutathione and H2O2. S-Glutathionylation of cysteine residues may introduce steric hindrance and allosteric effects to the active site. Collectively, these results provide detailed mechanistic insights into the substrate selectivity, redox regulation, and catalytic mechanism of PBCP.


Assuntos
Oryza/enzimologia , Monoéster Fosfórico Hidrolases/química , Proteínas de Plantas/química , Domínio Catalítico , Peróxido de Hidrogênio/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Fotossíntese , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Especificidade por Substrato
20.
FEBS Lett ; 593(2): 163-174, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30485416

RESUMO

In Photosystem II, loop E of the chlorophyll-binding CP47 protein is located near a redox-active tyrosine, YD , forming a symmetrical analog to loop E in CP43, which provides a ligand to the oxygen-evolving complex (OEC). A Glu364 to Gln substitution in CP47, near YD , does not affect growth in the cyanobacterium Synechocystis sp. PCC 6803; however, deletion of the extrinsic protein PsbV in this mutant leads to a strain displaying a pH-sensitive phenotype. Using thermoluminescence, chlorophyll fluorescence, and flash-induced oxygen evolution analyses, we demonstrate that Glu364 influences the stability of YD and the redox state of the OEC, and highlight the effects of external pH on photosynthetic electron transfer in intact cyanobacterial cells.


Assuntos
Substituição de Aminoácidos , Clorofila/metabolismo , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Complexos de Proteínas Captadores de Luz/genética , Modelos Moleculares , Oxirredução , Complexo de Proteína do Fotossistema II/genética , Ligação Proteica , Synechocystis/genética , Tirosina/metabolismo
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