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1.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445505

RESUMO

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme's sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle's genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an 'Fe-only' nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.


Assuntos
Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/metabolismo , Engenharia Genética/métodos , Nitrogenase/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Genoma de Cloroplastos , Fixação de Nitrogênio , Nitrogenase/metabolismo , Fotossíntese , Biologia Sintética
2.
Int J Mol Sci ; 22(15)2021 Jul 28.
Artigo em Inglês | MEDLINE | ID: mdl-34360850

RESUMO

Autophagy is a conserved degradation pathway for recycling damaged organelles and aberrant proteins, and its important roles in plant adaptation to nutrient starvation have been generally reported. Previous studies found that overexpression of autophagy-related (ATG) gene MdATG10 enhanced the autophagic activity in apple roots and promoted their salt tolerance. The MdATG10 expression was induced by nitrogen depletion condition in both leaves and roots of apple plants. This study aimed to investigate the differences in the growth and physiological status between wild type and MdATG10-overexpressing apple plants in response to nitrogen starvation. A hydroponic system containing different nitrogen levels was used. The study found that the reduction in growth and nitrogen concentrations in different tissues caused by nitrogen starvation was relieved by MdATG10 overexpression. Further studies demonstrated the increased root growth and the higher nitrogen absorption and assimilation ability of transgenic plants. These characteristics contributed to the increased uptake of limited nitrogen nutrients by transgenic plants, which also reduced the starvation damage to the chloroplasts. Therefore, the MdATG10-overexpressing apple plants could maintain higher photosynthetic ability and possess better growth under nitrogen starvation stress.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Malus/metabolismo , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Autofagia , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/crescimento & desenvolvimento , Estresse Fisiológico
3.
J Plant Physiol ; 264: 153487, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34358944

RESUMO

AtCYP38, a thylakoid lumen localized immunophilin, is found to be essential for photosystem II assembly and maintenance, but how AtCYP38 functions in chloroplast remains unknown. Based on previous functional studies and its crystal structure, we hypothesize that AtCYP38 should function via binding its targets or cofactors in the thylakoid lumen. To identify potential interacting proteins of AtCYP38, we first adopted ATTED-II and STRING web-tools, and found 12 proteins functionally related to AtCYP38. We then screened a yeast two-hybrid library including an Arabidopsis genome wide cDNA with different domain of AtCYP38, and five thylakoid lumen-localized targets were identified. In order to specifically search interacting proteins of AtCYP38 in the thylakoid lumen, we generated a yeast two-hybrid mini library including the thylakoid lumenal proteins and lumenal fractions of thylakoid membrane proteins, and we obtained six thylakoid membrane proteins and nine thylakoid lumenal proteins as interacting proteins of AtCYP38. The interactions between AtCYP38 and several potential targets were further confirmed via pull-down and co-immunoprecipitation assays. Together, a couple of new potential candidate interacting proteins of AtCYP38 were identified, and the results will lay a foundation for unveiling the regulatory mechanisms in photosynthesis by AtCYP38.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Ciclofilinas/metabolismo , Proteínas de Arabidopsis/fisiologia , Ciclofilinas/fisiologia , Imunoprecipitação , Complexo de Proteína do Fotossistema II/metabolismo , Domínios e Motivos de Interação entre Proteínas , Técnicas do Sistema de Duplo-Híbrido
4.
J Plant Physiol ; 264: 153470, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34274841

RESUMO

After an episode of heat stress plants retain a cellular "memory" of this event, a phenomenon known as thermomemory. This mechanism allows plants to better cope against a subsequent heat event. Thermomemory occurs through the persistence of heat shock proteins (HSPs) synthesized after the first "priming" event. Maintenance of this thermomemory comes at the cost to growth though, therefore it is vital that the memory is reset when no longer required. Recently, it has been reported that autophagy is important for resetting the thermomemory. It has also been shown recently that in response to heat, Arabidopsis displays an increase in chloroplast free calcium concentration which is partially dependent on calcium sensing receptor (CAS) protein. It is not known what the purpose of this heat-activated calcium signal is. Therefore, we compared downstream responses to heat in wild type (WT) and cas mutants, as the latter produce a reduced chloroplast calcium signal to heat. We found that after thermopriming the cas mutants displayed a greater biomass and a reduced level of the small heat shock protein HSP 17.6 degradation compared to WT. cas mutants did not show an increase in free amino acid levels after thermopriming, suggesting reduced autophagy. These results suggest that heat-induced chloroplast calcium elevation is a positive signal for resetting of the thermomemory.


Assuntos
Cálcio/metabolismo , Cloroplastos/metabolismo , Transdução de Sinais , Aminoácidos/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico
5.
Nat Commun ; 12(1): 4194, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234144

RESUMO

Photomorphogenesis, light-mediated development, is an essential feature of all terrestrial plants. While chloroplast development and brassinosteroid (BR) signaling are known players in photomorphogenesis, proteins that regulate both pathways have yet to be identified. Here we report that DE-ETIOLATION IN THE DARK AND YELLOWING IN THE LIGHT (DAY), a membrane protein containing DnaJ-like domain, plays a dual-role in photomorphogenesis by stabilizing the BR receptor, BRI1, as well as a key enzyme in chlorophyll biosynthesis, POR. DAY localizes to both the endomembrane and chloroplasts via its first transmembrane domain and chloroplast transit peptide, respectively, and interacts with BRI1 and POR in their respective subcellular compartments. Using genetic analysis, we show that DAY acts independently on BR signaling and chlorophyll biogenesis. Collectively, this work uncovers DAY as a factor that simultaneously regulates BR signaling and chloroplast development, revealing a key regulator of photomorphogenesis that acts across cell compartments.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Membrana/metabolismo , Morfogênese/fisiologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/metabolismo , Clorofila/biossíntese , Cloroplastos/metabolismo , Estiolamento/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/genética , Proteínas de Choque Térmico HSP40/isolamento & purificação , Luz , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/efeitos da radiação , Mutação , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Plântula/crescimento & desenvolvimento , Transdução de Sinais/fisiologia
6.
Plant Mol Biol ; 106(6): 521-531, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34224063

RESUMO

KEY MESSAGE: We characterize a functional lincRNA, XH123 in cotton seedling in defense of cold stress. The silencing of XH123 leads to increased sensitivity to cold stress and the decay of chloroplast. Cotton, which originated from the arid mid-American region, is one of the most important cash crops worldwide. Cultivated cotton is now widely spread throughout high-altitude regions such as those in the far northwest of Asia. In such areas, spring temperatures below 12 ℃ impose cold stress on cotton seedlings, with concomitant threat of lost yield and productivity. It is documented that cold stress can induce differential expression of long noncoding RNAs (lncRNAs) in cotton; however, it is not yet clear if these cold-responsive lncRNAs are actively involved with tolerance of cold stress at the molecular level. Here, we select ten long intergenic non-coding RNAs as candidate genes and use virus-induced gene silencing and additional cold treatments to examine their roles in the response to cold stress during the cotton seedling stage. One such gene, XH123, was revealed to be involved in tolerance of cold stress. Specifically, XH123-silenced plants demonstrated sensitivity to cold stress, exhibiting chloroplast damage and increased endogenous levels of reactive oxygen species. The transcriptome profile of XH123-silenced seedlings was similar to that of cold-stressed seedlings having the known cold stress gene PIF3 silenced. These results imply that the lincRNA XH123 is actively involved with cold stress regulation in cotton during the seedling stage.


Assuntos
Resposta ao Choque Frio/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica de Plantas , Gossypium/genética , RNA Longo não Codificante/genética , RNA de Plantas/genética , Adaptação Fisiológica/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Temperatura Baixa , Inativação Gênica , Gossypium/crescimento & desenvolvimento , Microscopia Eletrônica de Transmissão , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , RNA-Seq/métodos , Plântula/genética , Plântula/crescimento & desenvolvimento
7.
BMC Plant Biol ; 21(1): 342, 2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34281507

RESUMO

BACKGROUND: Chloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen (1O2) is unique in that it can signal to initiate cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsis thaliana mutant plastid ferrochelatase two (fc2) that conditionally accumulates chloroplast 1O2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently, it was demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1O2-stressed chloroplasts and cells has remained unexplored. RESULTS: To further understand 1O2-induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1O2-stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1O2 stress. Mutations affecting the core autophagy machinery (atg5, atg7, and atg10) were unable to suppress 1O2-induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1O2-mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1O2-damaged chloroplasts. CONCLUSIONS: Our results support the hypothesis that 1O2-dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1O2-damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1O2-induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Autofagia/genética , Morte Celular , Cloroplastos/metabolismo , Ferroquelatase/genética , Oxigênio Singlete/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Morte Celular/genética , Ferroquelatase/metabolismo , Genes de Plantas , Mutação , Plastídeos/metabolismo , Plântula , Estresse Fisiológico , Transcriptoma
8.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202438

RESUMO

The chloroplast is a semi-autonomous organelle with its own genome. The expression of chloroplast genes depends on both chloroplasts and the nucleus. Although many nucleus-encoded proteins have been shown to localize in chloroplasts and are essential for chloroplast gene expression, it is not clear whether transcription factors can regulate gene expression in chloroplasts. Here we report that the transcription factor NAC102 localizes in both chloroplasts and nucleus in Arabidopsis. Specifically, NAC102 localizes in chloroplast nucleoids. Yeast two-hybrid assay and co-immunoprecipitation assay suggested that NAC102 interacts with chloroplast RNA polymerases. Furthermore, overexpression of NAC102 in chloroplasts leads to reduced chloroplast gene expression and chlorophyll content, indicating that NAC102 functions as a repressor in chloroplasts. Our study not only revealed that transcription factors are new regulators of chloroplast gene expression, but also discovered that transcription factors can function in chloroplasts in addition to the canonical organelle nucleus.


Assuntos
Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Núcleo Celular , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação Proteica , Transporte Proteico
9.
Nucleic Acids Res ; 49(12): 6771-6787, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133716

RESUMO

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/genética , Proteínas de Ligação a DNA/metabolismo , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/enzimologia , Cloroplastos/metabolismo , Dano ao DNA , DNA de Plantas/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Instabilidade Genômica , Mutação , RNA de Plantas/metabolismo
10.
Nat Commun ; 12(1): 3685, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140516

RESUMO

Chloroplast NADH dehydrogenase-like (NDH) complex is structurally related to mitochondrial Complex I and forms a supercomplex with two copies of Photosystem I (the NDH-PSI supercomplex) via linker proteins Lhca5 and Lhca6. The latter was acquired relatively recently in a common ancestor of angiosperms. Here we show that NDH-dependent Cyclic Electron Flow 5 (NDF5) is an NDH assembly factor in Arabidopsis. NDF5 initiates the assembly of NDH subunits (PnsB2 and PnsB3) and Lhca6, suggesting that they form a contact site with Lhca6. Our analysis of the NDF5 ortholog in Physcomitrella and angiosperm genomes reveals the subunit PnsB2 to be newly acquired via tandem gene duplication of NDF5 at some point in the evolution of angiosperms. Another Lhca6 contact subunit, PnsB3, has evolved from a protein unrelated to NDH. The structure of the largest photosynthetic electron transport chain complex has become more complicated by acquiring novel subunits and supercomplex formation with PSI.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , NADH Desidrogenase/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bryopsida/genética , Evolução Molecular , Regulação da Expressão Gênica de Plantas/genética , Técnicas de Inativação de Genes , Hepatófitas/genética , Magnoliopsida/genética , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo
11.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064353

RESUMO

The lipid bilayer matrix of the thylakoid membrane of cyanobacteria and chloroplasts of plants and algae is mainly composed of uncharged galactolipids, but also contains anionic lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) as major constituents. The necessity of PG for photosynthesis is evident in all photosynthetic organisms examined to date, whereas the requirement of SQDG varies with species. In plants, although PG and SQDG are also found in non-photosynthetic plastids, their importance for the growth and functions of non-photosynthetic organs remains unclear. In addition, plants synthesize another anionic lipid glucuronosyldiacylglycerol (GlcADG) during phosphorus starvation, but its role in plant cells is not elucidated yet. To understand the functional relationships among PG, SQDG, and GlcADG, we characterized several Arabidopsis thaliana mutants defective in biosynthesis of these lipids. The mutants completely lacking both PG and SQDG biosynthesis in plastids showed developmental defects of roots, hypocotyls, and embryos in addition to leaves, which suggests that these lipids are pleiotropically required for the development of both photosynthetic and non-photosynthetic organs. Furthermore, our analysis revealed that SQDG, but not GlcADG, is essential for complementing the role of PG, particularly in photosynthesis under PG-deficient conditions such as phosphorus starvation.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Diglicerídeos/metabolismo , Glicolipídeos/metabolismo , Fosfatidilgliceróis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Galactolipídeos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Hipocótilo/citologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Mutação , Células Vegetais/metabolismo , Folhas de Planta/citologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Sementes/citologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
12.
Methods Mol Biol ; 2317: 77-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028763

RESUMO

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.


Assuntos
Biotecnologia/métodos , Cloroplastos/genética , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Transgenes , Animais , Cloroplastos/metabolismo , Humanos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo
13.
Methods Mol Biol ; 2317: 305-318, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028778

RESUMO

The green unicellular alga Chlamydomonas reinhardtii has emerged as a very attractive model system for chloroplast genetic engineering. Algae can be transformed readily at the chloroplast level through bombardment of cells with a gene gun and transformants can be selected using antibiotic resistance or phototrophic growth. An inducible chloroplast gene expression system could be very useful for several reasons. First, it could be used to elucidate the function of essential chloroplast genes required for cell growth and survival. Second, it could be very helpful for expressing proteins which are toxic to the algal cells. Third, it would allow for the reversible depletion of photosynthetic complexes, thus making it possible to study their biogenesis in a controlled fashion. Fourth, it opens promising possibilities for hydrogen production in Chlamydomonas. Here we describe an inducible/ repressible chloroplast gene expression system in Chlamydomonas in which the copper-regulated Cyc6 promoter or the vitamin-controlled MetE promoter and TPP riboswitch drive the expression of the nuclear Nac2 gene encoding a protein which is targeted to the chloroplast where it acts specifically on the chloroplast psbD 5' untranslated region and is required for the stable accumulation of the psbD mRNA and photosystem II. The system can be used for any chloroplast gene or trans-gene by placing it under the control of the psbD 5'untranslated region.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Cloroplastos/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Regiões Promotoras Genéticas , Riboswitch
14.
Gene ; 791: 145715, 2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-33984444

RESUMO

Leptodermis scabrida complex is one of the important components of genus Leptodermis, which is mainly distributed in the Himalaya Mountains. It includes species of L. gracilis, L. hirsutiflora, L. hirsutiflora var. ciliata, L. kumaonensis, L. pilosa var. acanthoclada and L. scabrida. However, species boundaries and relationships within this complex are unclear based on current morphological and molecular evidence. We sequenced 13 complete chloroplast (cp) genomes representing seven taxa of the complex and two non-Leptodermis scabrida complex taxa. After de novo assembly and annotation, we performed comparative genomic analysis. All cp genomes showed highly conserved structures, and the genome sizes ranged from 154,369 bp to 154,885 bp and possessed the same GC content (37.5%). A total of 113 unique genes were identified in each cp sample, including 79 protein coding genes, 30 tRNAs, and four rRNAs. Repeat sequences and SSRs were detected, showing great similarity among all taxa in this complex. Six highly variable regions, including trnS-trnG, rps2-rpoC2, ndhF, rpl32-ccsA, ccsA-ndhD, and ndhA, were screened as potential molecular markers for phylogenetic reconstruction. Based on a total of 27 complete cp genome sequences, the consistent and robust phylogenetic relationships were firstly constructed and the same species within L. scabrida complex clustered into a group. The divergence time of Leptodermis from ancestral taxa occurred at the middle Eocene, which might be due to geological and climatic changes. The 13 complete cp genome sequences reported will provide new clues for phylogeny elucidation, species identification and evolutionary history speculation of Leptodermis, as well as in Rubiaceae.


Assuntos
Genoma de Cloroplastos/genética , Rubiaceae/genética , Composição de Bases/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Evolução Molecular , Tamanho do Genoma/genética , Genômica/métodos , Repetições de Microssatélites/genética , Filogenia , RNA Ribossômico/genética , Rubiaceae/metabolismo , Sequenciamento Completo do Genoma
15.
New Phytol ; 231(4): 1431-1448, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33993494

RESUMO

Reactive oxygen species (ROS) produced in chloroplasts cause oxidative damage, but also signal to initiate chloroplast quality control pathways, cell death, and gene expression. The Arabidopsis thaliana plastid ferrochelatase two (fc2) mutant produces the ROS singlet oxygen in chloroplasts that activates such signaling pathways, but the mechanisms are largely unknown. Here we characterize one fc2 suppressor mutation and map it to CYTIDINE TRIPHOSPHATE SYNTHASE TWO (CTPS2), which encodes one of five enzymes in Arabidopsis necessary for de novo cytoplasmic CTP (and dCTP) synthesis. The ctps2 mutation reduces chloroplast transcripts and DNA content without similarly affecting mitochondria. Chloroplast nucleic acid content and singlet oxygen signaling are restored by exogenous feeding of the dCTP precursor deoxycytidine, suggesting ctps2 blocks signaling by limiting nucleotides for chloroplast genome maintenance. An investigation of CTPS orthologs in Brassicaceae showed CTPS2 is a member of an ancient lineage distinct from CTPS3. Complementation studies confirmed this analysis; CTPS3 was unable to compensate for CTPS2 function in providing nucleotides for chloroplast DNA and signaling. Our studies link cytoplasmic nucleotide metabolism with chloroplast quality control pathways. Such a connection is achieved by a conserved clade of CTPS enzymes that provide nucleotides for chloroplast function, thereby allowing stress signaling to occur.


Assuntos
Proteínas de Arabidopsis , Regulação da Expressão Gênica de Plantas , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Citidina Trifosfato , DNA de Cloroplastos/genética , Mutação , Nucleotídeos/genética
16.
J Exp Bot ; 72(13): 4949-4964, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-33963398

RESUMO

In plants, there is a complex interaction between carbon (C) and nitrogen (N) metabolism, and its coordination is fundamental for plant growth and development. Here, we studied the influence of thioredoxin (Trx) m on C and N partitioning using tobacco plants overexpressing Trx m from the chloroplast genome. The transgenic plants showed altered metabolism of C (lower leaf starch and soluble sugar accumulation) and N (with higher amounts of amino acids and soluble protein), which pointed to an activation of N metabolism at the expense of carbohydrates. To further delineate the effect of Trx m overexpression, metabolomic and enzymatic analyses were performed on these plants. These results showed an up-regulation of the glutamine synthetase-glutamate synthase pathway; specifically tobacco plants overexpressing Trx m displayed increased activity and stability of glutamine synthetase. Moreover, higher photorespiration and nitrate accumulation were observed in these plants relative to untransformed control plants, indicating that overexpression of Trx m favors the photorespiratory N cycle rather than primary nitrate assimilation. Taken together, our results reveal the importance of Trx m as a molecular mediator of N metabolism in plant chloroplasts.


Assuntos
Tiorredoxinas de Cloroplastos , Tabaco , Carbono/metabolismo , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
17.
Commun Biol ; 4(1): 545, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972654

RESUMO

Members of the pentatricopeptide repeat (PPR) protein family act as specificity factors in C-to-U RNA editing. The expansion of the PPR superfamily in plants provides the sequence variation required for design of consensus-based RNA-binding proteins. We used this approach to design a synthetic RNA editing factor to target one of the sites in the Arabidopsis chloroplast transcriptome recognised by the natural editing factor CHLOROPLAST BIOGENESIS 19 (CLB19). We show that our synthetic editing factor specifically recognises the target sequence in in vitro binding assays. The designed factor is equally specific for the target rpoA site when expressed in chloroplasts and in the bacterium E. coli. This study serves as a successful pilot into the design and application of programmable RNA editing factors based on plant PPR proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/genética , Escherichia coli/genética , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Cloroplastos/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética
18.
J Exp Bot ; 72(15): 5534-5552, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-33974689

RESUMO

In mature leaves, cell-to-cell transport via plasmodesmata between mesophyll cells links the production of assimilates by photosynthesis with their export to sink organs. This study addresses the question of how signals derived from chloroplasts and photosynthesis influence plasmodesmata permeability. Cell-to-cell transport was analyzed in leaves of the Arabidopsis chlorophyll b-less ch1-3 mutant, the same mutant complemented with a cyanobacterial CAO gene (PhCAO) overaccumulating chlorophyll b, the trxm3 mutant lacking plastidial thioredoxin m3, and the ntrc mutant lacking functional NADPH:thioredoxin reductase C. The regulation of plasmodesmata permeability in these lines could not be traced back to the reduction state of the thioredoxin system or the types and levels of reactive oxygen species produced in chloroplasts; however, it could be related to chloroplast ATP and NADPH production. The results suggest that light enables plasmodesmata closure via an increase in the ATP and NADPH levels produced in photosynthesis, providing a control mechanism for assimilate export based on the rate of photosynthate production in the Calvin-Benson cycle. The level of chlorophyll b influences plasmodesmata permeability via as-yet-unidentified signals. The data also suggest a role of thioredoxin m3 in the regulation of cyclic electron flow around photosystem I.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Trifosfato de Adenosina/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , NADP/metabolismo , Oxirredução , Fotossíntese , Folhas de Planta/metabolismo , Plasmodesmos/metabolismo
19.
J Exp Bot ; 72(15): 5553-5568, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-33989402

RESUMO

The kleptoplastic sea slug Elysia chlorotica consumes Vaucheria litorea, stealing its plastids, which then photosynthesize inside the animal cells for months. We investigated the properties of V. litorea plastids to understand how they withstand the rigors of photosynthesis in isolation. Transcription of specific genes in laboratory-isolated V. litorea plastids was monitored for 7 days. The involvement of plastid-encoded FtsH, a key plastid maintenance protease, in recovery from photoinhibition in V. litorea was estimated in cycloheximide-treated cells. In vitro comparison of V. litorea and spinach thylakoids was applied to investigate reactive oxygen species formation in V. litorea. In comparison to other tested genes, the transcripts of ftsH and translation elongation factor EF-Tu (tufA) decreased slowly in isolated V. litorea plastids. Higher levels of FtsH were also evident in cycloheximide-treated cells during recovery from photoinhibition. Charge recombination in PSII of V. litorea was found to be fine-tuned to produce only small quantities of singlet oxygen, and the plastids also contained reactive oxygen species-protective compounds. Our results support the view that the genetic characteristics of the plastids are crucial in creating a photosynthetic sea slug. The plastid's autonomous repair machinery is likely enhanced by low singlet oxygen production and elevated expression of FtsH.


Assuntos
Gastrópodes , Oxigênio Singlete , Animais , Cloroplastos/metabolismo , Gastrópodes/genética , Fotossíntese , Plastídeos , Oxigênio Singlete/metabolismo
20.
J Theor Biol ; 525: 110760, 2021 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-33984353

RESUMO

Many sea slugs of Sacoglossa (Mollusca: Heterobranchia) are sometimes called "solar-powered sea slugs" because they keep chloroplasts obtained from their food algae and receive photosynthetic products (termed kleptoplasty). Some species show life cycle dimorphism, in which a single species has some individuals with a complex life cycle (the mother produces planktotrophic larvae, which later settle in the adult habitat) and others with a simple life cycle (mothers produce benthic offspring by direct development or short-term nonfeeding larvae in which feeding planktonic stages are skipped). Life cycle dimorphism is not common among marine species. In this paper, we ask whether some aspects of the ecology of solar-powered sea slugs have promoted the evolution of life cycle dimorphism in them. We study the population dynamics of the two life-cycle types that differ in summer (one with planktonic life and the other with benthic life), but both have benthic life in other seasons. We obtain the conditions in which two types with different life cycles coexist stably or a single type generating offspring with different life cycles evolves. We conclude that the stable coexistence of two life cycles can evolve if benthic individuals in summer experience strongly density-dependent processes or if the between-year fluctuation of biomass growth in summer is very large. We discuss whether these results match the life cycles of solar-powered sea slugs with life cycle dimorphism.


Assuntos
Gastrópodes , Caracteres Sexuais , Animais , Cloroplastos/metabolismo , Larva , Fotossíntese
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