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1.
Plant Physiol ; 187(2): 931-946, 2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34608952

RESUMO

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

2.
New Phytol ; 230(3): 1258-1272, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33421132

RESUMO

CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.


Assuntos
Bryopsida , Bryopsida/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Mutagênese Sítio-Dirigida
3.
Nat Commun ; 12(1): 679, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514722

RESUMO

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.


Assuntos
Adaptação Fisiológica/genética , Proteínas de Ligação à Clorofila/metabolismo , Luz/efeitos adversos , Complexo de Proteína do Fotossistema I/metabolismo , Estramenópilas/fisiologia , Adaptação Fisiológica/efeitos da radiação , Clorofila A/metabolismo , Proteínas de Ligação à Clorofila/genética , Proteínas de Ligação à Clorofila/isolamento & purificação , Mutação , Fotossíntese/genética , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema I/genética , Estramenópilas/efeitos da radiação
4.
J Exp Bot ; 71(18): 5538-5548, 2020 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-32497206

RESUMO

Alternative electron pathways contribute to regulation of photosynthetic light reactions to adjust to metabolic demands in dynamic environments. The chloroplast NADH dehydrogenase-like (NDH) complex mediates the cyclic electron transport pathway around PSI in different cyanobacteria, algae, and plant species, but it is not fully conserved in all photosynthetic organisms. In order to assess how the physiological role of this complex changed during plant evolution, we isolated Physcomitrella patens lines knocked out for the NDHM gene that encodes a subunit fundamental for the activity of the complex. ndhm knockout mosses indicated high PSI acceptor side limitation upon abrupt changes in illumination. In P. patens, pseudo-cyclic electron transport mediated by flavodiiron proteins (FLVs) was also shown to prevent PSI over-reduction in plants exposed to light fluctuations. flva ndhm double knockout mosses had altered photosynthetic performance and growth defects under fluctuating light compared with the wild type and single knockout mutants. The results showed that while the contribution of NDH to electron transport is minor compared with FLV, NDH still participates in modulating photosynthetic activity, and it is critical to avoid PSI photoinhibition, especially when FLVs are inactive. The functional overlap between NDH- and FLV-dependent electron transport supports PSI activity and prevents its photoinhibition under light variations.


Assuntos
Bryopsida , Bryopsida/genética , Bryopsida/metabolismo , Cloroplastos/metabolismo , Transporte de Elétrons , Luz , NADH Desidrogenase/genética , NADH Desidrogenase/metabolismo , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo
5.
New Phytol ; 228(4): 1316-1326, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32367526

RESUMO

Photosynthetic electron transport is regulated by cyclic and pseudocyclic electron flow (CEF and PCEF) to maintain the balance between light availability and metabolic demands. CEF transfers electrons from photosystem I to the plastoquinone pool with two mechanisms, dependent either on PGR5/PGRL1 or on the type I NADH dehydrogenase-like (NDH) complex. PCEF uses electrons from photosystem I to reduce oxygen and in many groups of photosynthetic organisms, but remarkably not in angiosperms, it is catalyzed by flavodiiron proteins (FLVs). In this study, Physcomitrella patens plants depleted in PGRL1, NDH and FLVs in different combinations were generated and characterized, showing that all these mechanisms are active in this moss. Surprisingly, in contrast to flowering plants, Physcomitrella patens can cope with the simultaneous inactivation of PGR5- and NDH-dependent CEF but, when FLVs are also depleted, plants show strong growth reduction and photosynthetic activity is drastically reduced. The results demonstrate that mechanisms for modulation of photosynthetic electron transport have large functional overlap but are together indispensable to protect photosystem I from damage and they are an essential component for photosynthesis in any light regime.


Assuntos
Bryopsida , Complexo de Proteína do Fotossistema I , Bryopsida/metabolismo , Transporte de Elétrons , Luz , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Desenvolvimento Vegetal
6.
Sci Rep ; 10(1): 6770, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32317747

RESUMO

Although light is essential for photosynthesis, when in excess, it may damage the photosynthetic apparatus, leading to a phenomenon known as photoinhibition. Photoinhibition was thought as a light-induced damage to photosystem II; however, it is now clear that even photosystem I may become very vulnerable to light. One main characteristic of light induced damage to photosystem II (PSII) is the increased turnover of the reaction center protein, D1: when rate of degradation exceeds the rate of synthesis, loss of PSII activity is observed. With respect to photosystem I (PSI), an excess of electrons, instead of an excess of light, may be very dangerous. Plants possess a number of mechanisms able to prevent, or limit, such damages by safe thermal dissipation of light energy (non-photochemical quenching, NPQ), slowing-down of electron transfer through the intersystem transport chain (photosynthesis-control, PSC) in co-operation with the Proton Gradient Regulation (PGR) proteins, PGR5 and PGRL1, collectively called as short-term photoprotection mechanisms, and the redistribution of light between photosystems, called state transitions (responsible of fluorescence quenching at PSII, qT), is superimposed to these short term photoprotective mechanisms. In this manuscript we have generated a number of higher order mutants by crossing genotypes carrying defects in each of the short-term photoprotection mechanisms, with the final aim to obtain a direct comparison of their role and efficiency in photoprotection. We found that mutants carrying a defect in the ΔpH-dependent photosynthesis-control are characterized by photoinhibition of both photosystems, irrespectively of whether PSBS-dependent NPQ or state transitions defects were present or not in the same individual, demonstrating the primary role of PSC in photoprotection. Moreover, mutants with a limited capability to develop a strong PSBS-dependent NPQ, were characterized by a high turnover of the D1 protein and high values of Y(NO), which might reflect energy quenching processes occurring within the PSII reaction center.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Membrana/genética , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema II/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Genótipo , Concentração de Íons de Hidrogênio , Luz , Fotossíntese/genética , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema I/efeitos da radiação , Complexo de Proteína do Fotossistema II/efeitos da radiação
7.
Plant Cell Physiol ; 61(1): 41-52, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31511895

RESUMO

In nature, photosynthetic organisms are exposed to highly dynamic environmental conditions where the excitation energy and electron flow in the photosynthetic apparatus need to be continuously modulated. Fluctuations in incident light are particularly challenging because they drive oversaturation of photosynthesis with consequent oxidative stress and photoinhibition. Plants and algae have evolved several mechanisms to modulate their photosynthetic machinery to cope with light dynamics, such as thermal dissipation of excited chlorophyll states (non-photochemical quenching, NPQ) and regulation of electron transport. The regulatory mechanisms involved in the response to light dynamics have adapted during evolution, and exploring biodiversity is a valuable strategy for expanding our understanding of their biological roles. In this work, we investigated the response to fluctuating light in Nannochloropsis gaditana, a eukaryotic microalga of the phylum Heterokonta originating from a secondary endosymbiotic event. Nannochloropsis gaditana is negatively affected by light fluctuations, leading to large reductions in growth and photosynthetic electron transport. Exposure to light fluctuations specifically damages photosystem I, likely because of the ineffective regulation of electron transport in this species. The role of NPQ, also assessed using a mutant strain specifically depleted of this response, was instead found to be minor, especially in responding to the fastest light fluctuations.


Assuntos
Luz , Fotossíntese/fisiologia , Estramenópilas/metabolismo , Simbiose/fisiologia , Biodiversidade , Transporte de Elétrons/fisiologia , Estresse Oxidativo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema I/efeitos da radiação , Plantas/metabolismo , Estramenópilas/crescimento & desenvolvimento , Estramenópilas/efeitos da radiação
8.
Biochem J ; 476(17): 2487-2498, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519856

RESUMO

The regulation of photosynthesis is crucial to efficiently support the assimilation of carbon dioxide and to prevent photodamage. One key regulatory mechanism is the pseudo-cyclic electron flow (PCEF) mediated by class-C flavodiiron proteins (FLVs). These enzymes use electrons coming from Photosystem I (PSI) to reduce oxygen to water, preventing over-reduction in the acceptor side of PSI. FLVs are widely distributed among organisms performing oxygenic photosynthesis and they have been shown to be fundamental in many different conditions such as fluctuating light, sulfur deprivation and plant submersion. Moreover, since FLVs reduce oxygen they can help controlling the redox status of the cell and maintaining the microoxic environment essential for processes such as nitrogen fixation in cyanobacteria. Despite these important roles identified in various species, the genes encoding for FLV proteins have been lost in angiosperms where their activity could have been at least partially compensated by a more efficient cyclic electron flow (CEF). The present work reviews the information emerged on FLV function, analyzing recent structural data that suggest FLV could be regulated through a conformational change.


Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Fixação de Nitrogênio/fisiologia , Oxigênio/metabolismo , Fotossíntese/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Bactérias/genética , Cianobactérias/genética , Oxirredução , Complexo de Proteína do Fotossistema I/genética
9.
Photosynth Res ; 142(3): 249-264, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31270669

RESUMO

Non-photochemical quenching, NPQ, of chlorophyll fluorescence regulates the heat dissipation of chlorophyll excited states and determines the efficiency of the oxygenic photosynthetic systems. NPQ is regulated by a pH-sensing protein, responding to the chloroplast lumen acidification induced by excess light, coupled to an actuator, a chlorophyll/xanthophyll subunit where quenching reactions are catalyzed. In plants, the sensor is PSBS, while the two pigment-binding proteins Lhcb4 (also known as CP29) and LHCII are the actuators. In algae and mosses, stress-related light-harvesting proteins (LHCSR) comprise both functions of sensor and actuator within a single subunit. Here, we report on expressing the lhcsr1 gene from the moss Physcomitrella patens into several Arabidopsis thaliana npq4 mutants lacking the pH sensing PSBS protein essential for NPQ activity. The heterologous protein LHCSR1 accumulates in thylakoids of A. thaliana and NPQ activity can be partially restored. Complementation of double mutants lacking, besides PSBS, specific xanthophylls, allowed analyzing chromophore requirement for LHCSR-dependent quenching activity. We show that the partial recovery of NPQ is mostly due to the lower levels of Zeaxanthin in A. thaliana in comparison to P. patens. Complemented npq2npq4 mutants, lacking besides PSBS, Zeaxanthin Epoxidase, showed an NPQ recovery of up to 70% in comparison to A. thaliana wild type. Furthermore, we show that Lutein is not essential for the folding nor for the quenching activity of LHCSR1. In short, we have developed a system to study the function of LHCSR proteins using heterologous expression in a variety of A. thaliana mutants.


Assuntos
Arabidopsis/metabolismo , Bryopsida/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Luz , Complexos de Proteínas Captadores de Luz/genética , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Processos Fotoquímicos , Fotossíntese , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tilacoides/genética , Tilacoides/metabolismo , Xantofilas/metabolismo , Zeaxantinas/metabolismo
10.
Planta ; 249(4): 1217-1228, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30607502

RESUMO

MAIN CONCLUSION: Investigation of photosynthesis regulation in different plant groups exposed to variable conditions showed that all species have similar photosynthetic electron transport modulation while excess energy dissipation is species specific. Photosynthesis is regulated in response to dynamic environmental conditions to satisfy plant metabolic demands while also avoiding possible over-excitation of the electron transport chain and the generation of harmful reactive oxygen species. Photosynthetic organisms evolved several mechanisms to modulate light harvesting and electron transport efficiency to respond to conditions changing at different timescales, going from fast sun flecks to slow seasonal variations. These regulatory mechanisms changed during evolution of photosynthetic organisms, also adapting to various ecological niches, making the investigation of plant biodiversity highly valuable to uncover conserved traits and plasticity of photosynthetic regulation and complement studies on model species. In this work, a set of plants belonging to different genera of angiosperms, gymnosperms, ferns and lycophytes were investigated by monitoring their photosynthetic parameters in different seasons looking for common trends and differences. In all plants, analysed photosynthetic electron transport rate was found to be modulated by growth light intensity, ensuring a balance between available energy and photochemical capacity. Growth light also influenced the threshold where heat dissipation of excitation energy, a mechanism called non-photochemical quenching (NPQ), was activated. On the contrary, NPQ amplitude did not correlate with light intensity experienced by the plants but was a species-specific feature. The zeaxanthin-dependent component of NPQ, qZ, was found to be the most variable in different plants and its modulation influenced the intensity and the kinetic properties of the response.


Assuntos
Biodiversidade , Fotossíntese/fisiologia , Plantas/metabolismo , Transporte de Elétrons , Meio Ambiente , Luz , Complexo de Proteína do Fotossistema II/metabolismo
11.
New Phytol ; 221(1): 105-109, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30084195

RESUMO

Contents Summary 105 I. Introduction 105 II. Diversity of molecular mechanisms for regulation of photosynthetic electron transport 106 III. Role of FLVs in the regulation of photosynthesis in eukaryotes 107 IV. Why were FLVs lost in angiosperms? 108 V. Conclusions 108 Acknowledgements 109 References 109 SUMMARY: Photosynthetic electron transport requires continuous modulation to maintain the balance between light availability and metabolic demands. Multiple mechanisms for the regulation of electron transport have been identified and are unevenly distributed among photosynthetic organisms. Flavodiiron proteins (FLVs) influence photosynthetic electron transport by accepting electrons downstream of photosystem I to reduce oxygen to water. FLV activity has been demonstrated in cyanobacteria, green algae and mosses to be important in avoiding photosystem I overreduction upon changes in light intensity. FLV-encoding sequences were nevertheless lost during evolution by angiosperms, suggesting that these plants increased the efficiency of other mechanisms capable of accepting electrons from photosystem I, making the FLV activity for protection from overreduction superfluous or even detrimental for photosynthetic efficiency.


Assuntos
Evolução Biológica , Transporte de Elétrons , Fotossíntese/fisiologia , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/metabolismo , Magnoliopsida/fisiologia , Complexo de Proteína do Fotossistema I/metabolismo
12.
Plant Cell Environ ; 42(5): 1590-1602, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30496624

RESUMO

Photosynthetic organisms support cell metabolism by harvesting sunlight and driving the electron transport chain at the level of thylakoid membranes. Excitation energy and electron flow in the photosynthetic apparatus is continuously modulated in response to dynamic environmental conditions. Alternative electron flow around photosystem I plays a seminal role in this regulation contributing to photoprotection by mitigating overreduction of the electron carriers. Different pathways of alternative electron flow coexist in the moss Physcomitrella patens, including cyclic electron flow mediated by the PGRL1/PGR5 complex and pseudo-cyclic electron flow mediated by the flavodiiron proteins FLV. In this work, we generated P. patens plants carrying both pgrl1 and flva knock-out mutations. A comparative analysis of the WT, pgrl1, flva, and pgrl1 flva lines suggests that cyclic and pseudo-cyclic processes have a synergic role in the regulation of photosynthetic electron transport. However, although both contribute to photosystem I protection from overreduction by modulating electron flow following changes in environmental conditions, FLV activity is particularly relevant in the first seconds after a light change whereas PGRL1 has a major role upon sustained strong illumination.


Assuntos
Bryopsida/fisiologia , Transporte de Elétrons/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteína do Fotossistema I/metabolismo , Bryopsida/genética , Cloroplastos/metabolismo , Transporte de Elétrons/genética , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fotossíntese/genética , Fotossíntese/fisiologia , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema I/genética , Plantas Geneticamente Modificadas , Luz Solar , Tilacoides/metabolismo
13.
Nat Plants ; 4(11): 910-919, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30374091

RESUMO

Photosystem I of the moss Physcomitrella patens has special properties, including the capacity to undergo non-photochemical fluorescence quenching. We studied the organization of photosystem I under different light and carbon supply conditions in wild-type moss and in moss with the lhcb9 (light-harvesting complex) knockout genotype, which lacks an antenna protein endowed with red-shifted absorption forms. Wild-type moss, when grown on sugars and in low light, accumulated LHCB9 proteins and a large form of the photosystem I supercomplex, which, besides the canonical four LHCI subunits, included a LHCII trimer and four additional LHC monomers. The lhcb9 knockout produced an angiosperm-like photosystem I supercomplex with four LHCI subunits irrespective of the growth conditions. Growth in the presence of sublethal concentrations of electron transport inhibitors that caused oxidation or reduction of the plastoquinone pool prevented or promoted, respectively, the accumulation of LHCB9 and the formation of the photosystem I megacomplex. We suggest that LHCB9 is a key subunit regulating the antenna size of photosystem I and the ability to avoid the over-reduction of plastoquinone: this condition is potentially dangerous in the shaded and sunfleck-rich environment typical of mosses, whose plastoquinone pool is reduced by both photosystem II and the oxidation of sugar substrates.


Assuntos
Bryopsida/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Bryopsida/efeitos da radiação , Luz , Complexos de Proteínas Captadores de Luz/efeitos da radiação , Complexos de Proteínas Captadores de Luz/ultraestrutura , Microscopia Eletrônica , Complexo de Proteína do Fotossistema I/efeitos da radiação , Complexo de Proteína do Fotossistema I/ultraestrutura , Complexo de Proteína do Fotossistema II/metabolismo , Proteômica , Tilacoides/metabolismo
14.
Plant Cell Physiol ; 59(7): 1377-1384, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878186

RESUMO

The adaptation to dehydration and rehydration cycles represents a key step in the evolution of photosynthetic organisms and requires the development of mechanisms by which to sense external stimuli and translate them into signaling components. In this study, we used genetically encoded fluorescent sensors to detect specific transient increases in the Ca2+ concentration in the moss Physcomitrella patens upon dehydration and rehydration treatment. Observation of the entire plant in a single time-series acquisition revealed that various cell types exhibited different sensitivities to osmotic stress and that Ca2+ waves originated from the basal part of the gametophore and were directionally propagated towards the top of the plant. Under similar conditions, the vascular plant Arabidopsis thaliana exhibited Ca2+ waves that propagated at a higher speed than those of P. patens. Our results suggest that systemic Ca2+ propagation occurs in plants even in the absence of vascular tissue, even though the rates can be different.


Assuntos
Bryopsida/metabolismo , Sinalização do Cálcio , Arabidopsis/metabolismo , Bryopsida/citologia , Bryopsida/fisiologia , Cálcio/análise , Cálcio/metabolismo , Calmodulina/metabolismo , Desidratação , Transferência Ressonante de Energia de Fluorescência , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Pressão Osmótica , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/metabolismo
15.
Plant J ; 95(1): 168-182, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29681058

RESUMO

High-throughput RNA sequencing (RNA-seq) has recently become the method of choice to define and analyze transcriptomes. For the model moss Physcomitrella patens, although this method has been used to help analyze specific perturbations, no overall reference dataset has yet been established. In the framework of the Gene Atlas project, the Joint Genome Institute selected P. patens as a flagship genome, opening the way to generate the first comprehensive transcriptome dataset for this moss. The first round of sequencing described here is composed of 99 independent libraries spanning 34 different developmental stages and conditions. Upon dataset quality control and processing through read mapping, 28 509 of the 34 361 v3.3 gene models (83%) were detected to be expressed across the samples. Differentially expressed genes (DEGs) were calculated across the dataset to permit perturbation comparisons between conditions. The analysis of the three most distinct and abundant P. patens growth stages - protonema, gametophore and sporophyte - allowed us to define both general transcriptional patterns and stage-specific transcripts. As an example of variation of physico-chemical growth conditions, we detail here the impact of ammonium supplementation under standard growth conditions on the protonemal transcriptome. Finally, the cooperative nature of this project allowed us to analyze inter-laboratory variation, as 13 different laboratories around the world provided samples. We compare differences in the replication of experiments in a single laboratory and between different laboratories.


Assuntos
Bryopsida/genética , Conjuntos de Dados como Assunto , Genes de Plantas/genética , Mapeamento Cromossômico , Genoma de Planta/genética , Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma/genética
16.
New Phytol ; 214(3): 967-972, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28304077

RESUMO

Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.


Assuntos
Cianobactérias/metabolismo , Cycadopsida/metabolismo , Flavoproteínas/metabolismo , Cianobactérias/efeitos da radiação , Cycadopsida/efeitos da radiação , Transporte de Elétrons , Cinética , Luz , Oxirredução , Fotossíntese/efeitos da radiação , Filogenia
17.
New Phytol ; 213(2): 714-726, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27620972

RESUMO

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained.


Assuntos
Sequência Conservada , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/metabolismo , Subunidades Proteicas/metabolismo , Estramenópilas/metabolismo , Simbiose , Sequência de Aminoácidos , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Complexos de Proteínas Captadores de Luz/ultraestrutura , Modelos Biológicos , Complexo de Proteína do Fotossistema I/ultraestrutura , Pigmentos Biológicos/metabolismo , Subunidades Proteicas/química , Espectrometria de Fluorescência , Tilacoides/metabolismo
18.
Proc Natl Acad Sci U S A ; 113(43): 12322-12327, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27791022

RESUMO

Photosynthetic organisms support cell metabolism by harvesting sunlight to fuel the photosynthetic electron transport. The flow of excitation energy and electrons in the photosynthetic apparatus needs to be continuously modulated to respond to dynamics of environmental conditions, and Flavodiiron (FLV) proteins are seminal components of this regulatory machinery in cyanobacteria. FLVs were lost during evolution by flowering plants, but are still present in nonvascular plants such as Physcomitrella patens We generated P. patens mutants depleted in FLV proteins, showing their function as an electron sink downstream of photosystem I for the first seconds after a change in light intensity. flv knock-out plants showed impaired growth and photosystem I photoinhibition when exposed to fluctuating light, demonstrating FLV's biological role as a safety valve from excess electrons on illumination changes. The lack of FLVs was partially compensated for by an increased cyclic electron transport, suggesting that in flowering plants, the FLV's role was taken by other alternative electron routes.


Assuntos
Bryopsida/genética , Evolução Molecular , Fotossíntese/genética , Proteínas de Plantas/genética , Bryopsida/crescimento & desenvolvimento , Transporte de Elétrons/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oxigênio/metabolismo , Luz Solar
19.
Biophys Chem ; 218: 14-26, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27586818

RESUMO

Photosynthetic eukaryotes have a complex evolutionary history shaped by multiple endosymbiosis events that required a tight coordination between the organelles and the rest of the cell. Plant ionotropic glutamate receptors (iGLRs) form a large superfamily of proteins with a predicted or proven non-selective cation channel activity regulated by a broad range of amino acids. They are involved in different physiological processes such as C/N sensing, resistance against fungal infection, root and pollen tube growth and response to wounding and pathogens. Most of the present knowledge is limited to iGLRs located in plasma membranes. However, recent studies localized different iGLR isoforms to mitochondria and/or chloroplasts, suggesting the possibility that they play a specific role in bioenergetic processes. In this work, we performed a comparative analysis of GLR sequences from bacteria and various photosynthetic eukaryotes. In particular, novel types of selectivity filters of bacteria are reported adding new examples of the great diversity of the GLR superfamily. The highest variability in GLR sequences was found among the algal sequences (cryptophytes, diatoms, brown and green algae). GLRs of land plants are not closely related to the GLRs of green algae analyzed in this work. The GLR family underwent a great expansion in vascular plants. Among plant GLRs, Clade III includes sequences from Physcomitrella patens, Marchantia polymorpha and gymnosperms and can be considered the most ancient, while other clades likely emerged later. In silico analysis allowed the identification of sequences with a putative target to organelles. Sequences with a predicted localization to mitochondria and chloroplasts are randomly distributed among different type of GLRs, suggesting that no compartment-related specific function has been maintained across the species.


Assuntos
Evolução Molecular , Filogenia , Receptores Ionotrópicos de Glutamato/genética , Sequência de Aminoácidos , Animais , Clorófitas/química , Cloroplastos/química , Embriófitas/química , Mitocôndrias/química , Fotossíntese/genética , Alinhamento de Sequência
20.
Plant Physiol ; 171(4): 2468-82, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27325666

RESUMO

The seawater microalga Nannochloropsis gaditana is capable of accumulating a large fraction of reduced carbon as lipids. To clarify the molecular bases of this metabolic feature, we investigated light-driven lipid biosynthesis in Nannochloropsis gaditana cultures combining the analysis of photosynthetic functionality with transcriptomic, lipidomic and metabolomic approaches. Light-dependent alterations are observed in amino acid, isoprenoid, nucleic acid, and vitamin biosynthesis, suggesting a deep remodeling in the microalgal metabolism triggered by photoadaptation. In particular, high light intensity is shown to affect lipid biosynthesis, inducing the accumulation of diacylglyceryl-N,N,N-trimethylhomo-Ser and triacylglycerols, together with the up-regulation of genes involved in their biosynthesis. Chloroplast polar lipids are instead decreased. This situation correlates with the induction of genes coding for a putative cytosolic fatty acid synthase of type 1 (FAS1) and polyketide synthase (PKS) and the down-regulation of the chloroplast fatty acid synthase of type 2 (FAS2). Lipid accumulation is accompanied by the regulation of triose phosphate/inorganic phosphate transport across the chloroplast membranes, tuning the carbon metabolic allocation between cell compartments, favoring the cytoplasm, mitochondrion, and endoplasmic reticulum at the expense of the chloroplast. These results highlight the high flexibility of lipid biosynthesis in N. gaditana and lay the foundations for a hypothetical mechanism of regulation of primary carbon partitioning by controlling metabolite allocation at the subcellular level.


Assuntos
Carbono/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Metabolismo dos Lipídeos/efeitos da radiação , Fotossíntese/efeitos da radiação , Estramenópilas/metabolismo , Cloroplastos/metabolismo , Cloroplastos/efeitos da radiação , Regulação para Baixo/efeitos da radiação , Luz , Microalgas , Estramenópilas/efeitos da radiação , Triglicerídeos/metabolismo , Regulação para Cima/efeitos da radiação
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