RESUMEN
The initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis for most life on Earth, is catalyzed by photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we used Arabidopsis (Arabidopsis thaliana) as a model to show that in plants, a eukaryote-exclusive assembly factor facilitates the early assembly step, during which the intrinsic antenna protein CP47 becomes associated with the PSII reaction center (RC) to form the RC47 intermediate. This factor, which we named DECREASED ELECTRON TRANSPORT AT PSII (DEAP2), works in concert with the conserved PHOTOSYNTHESIS AFFECTED MUTANT 68 (PAM68) assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins resulted in a loss of functional PSII and the inability of plants to grow photoautotrophically on the soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulated at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Complejo de Proteína del Fotosistema II/genética , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Eucariontes/metabolismo , Fotosíntesis , Plantas/metabolismoRESUMEN
Genetic incompatibility between the cytoplasm and the nucleus is thought to be a major factor in species formation, but mechanistic understanding of this process is poor. In evening primroses (Oenothera spp.), a model plant for organelle genetics and population biology, hybrid offspring regularly display chloroplast-nuclear incompatibility. This usually manifests in bleached plants, more rarely in hybrid sterility or embryonic lethality. Hence, most of these incompatibilities affect photosynthetic capability, a trait that is under selection in changing environments. Here we show that light-dependent misregulation of the plastid psbB operon, which encodes core subunits of photosystem II and the cytochrome b6f complex, can lead to hybrid incompatibility, and this ultimately drives speciation. This misregulation causes an impaired light acclimation response in incompatible plants. Moreover, as a result of their different chloroplast genotypes, the parental lines differ in photosynthesis performance upon exposure to different light conditions. Significantly, the incompatible chloroplast genome is naturally found in xeric habitats with high light intensities, whereas the compatible one is limited to mesic habitats. Consequently, our data raise the possibility that the hybridization barrier evolved as a result of adaptation to specific climatic conditions.
Asunto(s)
Especiación Genética , Genoma del Cloroplasto , Oenothera biennis/genética , Operón , Fotosíntesis/genética , Aclimatación/genética , Complejo de Citocromo b6f/genética , Luz , Oenothera biennis/fisiología , Complejo de Proteína del Fotosistema II/genética , Proteínas de Plantas/genética , Plastidios/genética , Regiones Promotoras Genéticas , Edición de ARNRESUMEN
Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.
Asunto(s)
Cloroplastos/metabolismo , Mutación del Sistema de Lectura/genética , Genes de Plantas , Nicotiana/genética , Oenothera/genética , Proteínas de Plantas/genética , Biosíntesis de Proteínas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cloroplastos/ultraestructura , ADN Complementario/genética , Escherichia coli/metabolismo , Genotipo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Péptidos/química , Péptidos/metabolismo , Fenotipo , Fotosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , ReproducciónRESUMEN
RNA editing converts cytidines to uridines in plant organellar transcripts. Editing typically restores codons for conserved amino acids. During evolution, specific C-to-U editing sites can be lost from some plant lineages by genomic C-to-T mutations. By contrast, the emergence of novel editing sites is less well documented. Editing sites are recognized by pentatricopeptide repeat (PPR) proteins with high specificity. RNA recognition by PPR proteins is partially predictable, but prediction is often inadequate for PPRs involved in RNA editing. Here we have characterized evolution and recognition of a recently gained editing site. We demonstrate that changes in the RNA recognition motifs that are not explainable with the current PPR code allow an ancient PPR protein, QED1, to uniquely target the ndhB-291 site in Brassicaceae. When expressed in tobacco, the Arabidopsis QED1 edits 33 high-confident off-target sites in chloroplasts and mitochondria causing a spectrum of mutant phenotypes. By manipulating the relative expression levels of QED1 and ndhB-291, we show that the target specificity of the PPR protein depends on the RNA:protein ratio. Finally, our data suggest that the low expression levels of PPR proteins are necessary to ensure the specificity of editing site selection and prevent deleterious off-target editing.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Edición de ARN , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , ARN , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
The pathway of photosystem II (PSII) assembly is well understood, and multiple auxiliary proteins supporting it have been identified, but little is known about rate-limiting steps controlling PSII biogenesis. In the cyanobacterium Synechocystis PCC6803 and the green alga Chlamydomonas reinhardtii, indications exist that the biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, we modified the 5'-untranslated region of psbD via chloroplast transformation in tobacco (Nicotiana tabacum). A drastic reduction in psbD mRNA abundance resulted in a strong decrease in PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in cyanobacteria or in C. reinhardtii.
Asunto(s)
Nicotiana/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , ARN Mensajero/metabolismo , Cloroplastos/genética , Cloroplastos/metabolismo , Complejo de Proteína del Fotosistema II/genética , Biosíntesis de Proteínas/genética , Biosíntesis de Proteínas/fisiología , Nicotiana/genéticaRESUMEN
During photosynthesis, energy is transiently stored as an electrochemical proton gradient across the thylakoid membrane. The resulting proton motive force (pmf) is composed of a membrane potential (ΔΨ) and a proton concentration gradient (ΔpH) and powers the synthesis of ATP. Light energy availability for photosynthesis can change very rapidly and frequently in nature. Thylakoid ion transport proteins buffer the effects that light fluctuations have on photosynthesis by adjusting pmf and its composition. Ion channel activities dissipate ΔΨ, thereby reducing charge recombinations within photosystem II. The dissipation of ΔΨ allows for increased accumulation of protons in the thylakoid lumen, generating the signal that activates feedback downregulation of photosynthesis. Proton export from the lumen via the thylakoid K+ exchange antiporter 3 (KEA3), instead, decreases the ΔpH fraction of the pmf and thereby reduces the regulatory feedback signal. Here, we reveal that the Arabidopsis (Arabidopsis thaliana) KEA3 protein homo-dimerizes via its C-terminal domain. This C-terminus has a regulatory function, which responds to light intensity transients. Plants carrying a C-terminus-less KEA3 variant show reduced feed-back downregulation of photosynthesis and suffer from increased photosystem damage under long-term high light stress. However, during photosynthetic induction in high light, KEA3 deregulation leads to an increase in carbon fixation rates. Together, the data reveal a trade-off between long-term photoprotection and a short-term boost in carbon fixation rates, which is under the control of the KEA3 C-terminus.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Tilacoides/metabolismoRESUMEN
During photosynthesis, electrons travel from light-excited chlorophyll molecules along the electron transport chain to the final electron acceptor nicotinamide adenine dinucleotide phosphate (NADP) to form NADPH, which fuels the Calvin-Benson-Bassham cycle (CBBC). To allow photosynthetic reactions to occur flawlessly, a constant resupply of the acceptor NADP is mandatory. Several known stromal mechanisms aid in balancing the redox poise, but none of them utilizes the structurally highly similar coenzyme NAD(H). Using Arabidopsis (Arabidopsis thaliana) as a C3-model, we describe a pathway that employs the stromal enzyme PHOSPHOGLYCERATE DEHYDROGENASE 3 (PGDH3). We showed that PGDH3 exerts high NAD(H)-specificity and is active in photosynthesizing chloroplasts. PGDH3 withdrew its substrate 3-PGA directly from the CBBC. As a result, electrons become diverted from NADPH via the CBBC into the separate NADH redox pool. pgdh3 loss-of-function mutants revealed an overreduced NADP(H) redox pool but a more oxidized plastid NAD(H) pool compared to wild-type plants. As a result, photosystem I acceptor side limitation increased in pgdh3. Furthermore, pgdh3 plants displayed delayed CBBC activation, changes in nonphotochemical quenching, and altered proton motive force partitioning. Our fluctuating light-stress phenotyping data showed progressing photosystem II damage in pgdh3 mutants, emphasizing the significance of PGDH3 for plant performance under natural light environments. In summary, this study reveals an NAD(H)-specific mechanism in the stroma that aids in balancing the chloroplast redox poise. Consequently, the stromal NAD(H) pool may provide a promising target to manipulate plant photosynthesis.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , NAD , Fosfoglicerato-Deshidrogenasa , Fotosíntesis , Arabidopsis/enzimología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , NAD/metabolismo , Fosfoglicerato-Deshidrogenasa/metabolismoRESUMEN
In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast-replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate-limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turnover rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, as plastid competitiveness can result in uniparental inheritance (through elimination of the "weak" plastid) or biparental inheritance (when two similarly "strong" plastids are transmitted).
Asunto(s)
Cloroplastos/genética , Cloroplastos/fisiología , Oenothera biennis/metabolismo , Acetil-CoA Carboxilasa/genética , Evolución Biológica , Núcleo Celular/genética , Citoplasma/genética , Eucariontes/genética , Genoma , Genoma de Plastidios/genética , Genotipo , Lípidos/biosíntesis , Oenothera biennis/fisiología , Proteínas de Plantas/genética , Plastidios/genéticaRESUMEN
Because carotenoids act as accessory pigments in photosynthesis, play a key photoprotective role and are of major nutritional importance, carotenogenesis has been a target for crop improvement. Although carotenoids are important precursors of phytohormones, previous genetic manipulations reported little if any effects on biomass production and plant development, but resulted in specific modifications in carotenoid content. Unexpectedly, the expression of the carrot lycopene ß-cyclase (DcLCYB1) in Nicotiana tabacum cv. Xanthi not only resulted in increased carotenoid accumulation, but also in altered plant architecture characterized by longer internodes, faster plant growth, early flowering and increased biomass. Here, we have challenged these transformants with a range of growth conditions to determine the robustness of their phenotype and analyze the underlying mechanisms. Transgenic DcLCYB1 lines showed increased transcript levels of key genes involved in carotenoid, chlorophyll, gibberellin (GA) and abscisic acid (ABA) biosynthesis, but also in photosynthesis-related genes. Accordingly, their carotenoid, chlorophyll, ABA and GA contents were increased. Hormone application and inhibitor experiments confirmed the key role of altered GA/ABA contents in the growth phenotype. Because the longer internodes reduce shading of mature leaves, induction of leaf senescence was delayed, and mature leaves maintained a high photosynthetic capacity. This increased total plant assimilation, as reflected in higher plant yields under both fully controlled constant and fluctuating light, and in non-controlled conditions. Furthermore, our data are a warning that engineering of isoprenoid metabolism can cause complex changes in phytohormone homeostasis and therefore plant development, which have not been sufficiently considered in previous studies.
Asunto(s)
Carotenoides/metabolismo , Genes de Plantas/fisiología , Nicotiana/crecimiento & desarrollo , Fotosíntesis/genética , Ácido Abscísico/metabolismo , Daucus carota/genética , Regulación de la Expresión Génica de las Plantas/genética , Genes de Plantas/genética , Giberelinas/metabolismo , Redes y Vías Metabólicas/genética , Fotosíntesis/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Plantas Modificadas Genéticamente , Nicotiana/anatomía & histología , Nicotiana/metabolismo , Nicotiana/fisiología , Regulación hacia ArribaRESUMEN
Acclimation to changing light intensities poses major challenges to plant metabolism and has been shown to involve regulatory adjustments in chloroplast gene expression. However, this regulation has not been examined at a plastid genome-wide level and for many genes, it is unknown whether their expression responds to altered light intensities. Here, we applied comparative ribosome profiling and transcriptomic experiments to analyze changes in chloroplast transcript accumulation and translation in leaves of tobacco (Nicotiana tabacum) seedlings after transfer from moderate light to physiological high light. Our time-course data revealed almost unaltered chloroplast transcript levels and only mild changes in ribosome occupancy during 2 d of high light exposure. Ribosome occupancy on the psbA mRNA (encoding the D1 reaction center protein of PSII) increased and that on the petG transcript decreased slightly after high light treatment. Transfer from moderate light to high light did not induce substantial alterations in ribosome pausing. Transfer experiments from low light to high light conditions resulted in strong PSII photoinhibition and revealed the distinct light-induced activation of psbA translation, which was further confirmed by reciprocal shift experiments. In low-light-to-high-light shift experiments, as well as reciprocal treatments, the expression of all other chloroplast genes remained virtually unaltered. Altogether, our data suggest that low light-acclimated plants upregulate the translation of a single chloroplast gene, psbA, during acclimation to high light. Our results indicate that psbA translation activation occurs already at moderate light intensities. Possible reasons for the otherwise mild effects of light intensity changes on gene expression in differentiated chloroplasts are discussed.
Asunto(s)
Cloroplastos/metabolismo , Luz , Nicotiana/metabolismo , Cloroplastos/efectos de la radiación , Complejo de Proteína del Fotosistema II/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Ribosomas/efectos de la radiación , Nicotiana/efectos de la radiaciónRESUMEN
The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , ATPasas de Translocación de Protón de Cloroplastos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , ATPasas de Translocación de Protón de Cloroplastos/genética , Concentración de Iones de Hidrógeno , Fotosíntesis/genética , Fotosíntesis/fisiología , Complejo de Proteína del Fotosistema II/metabolismo , Antiportadores de Potasio-Hidrógeno/genética , Antiportadores de Potasio-Hidrógeno/metabolismo , Proteínas de las Membranas de los Tilacoides/genética , Proteínas de las Membranas de los Tilacoides/metabolismo , Tilacoides/metabolismoRESUMEN
Carotenoids are important isoprenoids produced in the plastids of photosynthetic organisms that play key roles in photoprotection and antioxidative processes. ß-Carotene is generated from lycopene by lycopene ß-cyclase (LCYB). Previously, we demonstrated that the introduction of the Daucus carota (carrot) DcLCYB1 gene into tobacco (cv. Xanthi) resulted in increased levels of abscisic acid (ABA) and especially gibberellins (GAs), resulting in increased plant yield. In order to understand this phenomenon prior to exporting this genetic strategy to crops, we generated tobacco (Nicotiana tabacum cv. Petit Havana) mutants that exhibited a wide range of LCYB expression. Transplastomic plants expressing DcLCYB1 at high levels showed a wild-type-like growth, even though their pigment content was increased and their leaf GA1 content was reduced. RNA interference (RNAi) NtLCYB lines showed different reductions in NtLCYB transcript abundance, correlating with reduced pigment content and plant variegation. Photosynthesis (leaf absorptance, Fv/Fm, and light-saturated capacity of linear electron transport) and plant growth were impaired. Remarkably, drastic changes in phytohormone content also occurred in the RNAi lines. However, external application of phytohormones was not sufficient to rescue these phenotypes, suggesting that altered photosynthetic efficiency might be another important factor explaining their reduced biomass. These results show that LCYB expression influences plant biomass by different mechanisms and suggests thresholds for LCYB expression levels that might be beneficial or detrimental for plant growth.
Asunto(s)
Liasas Intramoleculares , Nicotiana , Carotenoides , Regulación de la Expresión Génica de las Plantas , Liasas Intramoleculares/genética , Liasas Intramoleculares/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
In chloroplasts and plant mitochondria, specific cytidines in mRNAs are posttranscriptionally converted to uridines by RNA editing. Editing sites are recognized by nucleus-encoded RNA-binding proteins of the pentatricopeptide repeat (PPR) family, which bind upstream of the editing site in a sequence-specific manner and direct the editing activity to the target position. Editing sites have been lost many times during evolution by C-to-T mutations. Loss of an editing site is thought to be accompanied by loss or degeneration of its cognate PPR protein. Consequently, foreign editing sites are usually not recognized when introduced into species lacking the site. Previously, the spinach (Spinacia oleracea) psbF-26 editing site was introduced into the tobacco (Nicotiana tabacum) plastid genome. Tobacco lacks the psbF-26 site and cannot edit it. Expression of the "unedited" PsbF protein resulted in impaired PSII function. In Arabidopsis (Arabidopsis thaliana), the PPR protein LPA66 is required for editing at psbF-26. Here, we show that introduction of the Arabidopsis LPA66 reconstitutes editing of the spinach psbF-26 site in tobacco and restores a wild-type-like phenotype. Our findings define the minimum requirements for establishing new RNA editing sites and suggest that the evolutionary dynamics of editing patterns is largely explained by coevolution of editing sites and PPR proteins.
Asunto(s)
Arabidopsis/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Edición de ARN/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plastidios/genética , Plastidios/metabolismoRESUMEN
Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.
Asunto(s)
Nicotiana/citología , Hojas de la Planta/citología , Hojas de la Planta/fisiología , Biología de Sistemas/métodos , Aminoácidos/metabolismo , Metabolismo de los Hidratos de Carbono , Núcleo Celular/genética , Cloroplastos , Genoma de Plastidios , Luz , Metabolismo de los Lípidos , Microscopía Electrónica de Transmisión , Fotosíntesis , Plastidios/genética , Nicotiana/fisiología , Transcriptoma , Triglicéridos/metabolismoRESUMEN
Vegetative growth requires the systemic coordination of numerous cellular processes, which are controlled by regulatory proteins that monitor extracellular and intracellular cues and translate them into growth decisions. In eukaryotes, one of the central factors regulating growth is the serine/threonine protein kinase Target of Rapamycin (TOR), which forms complexes with regulatory proteins. To understand the function of one such regulatory protein, Regulatory-Associated Protein of TOR 1B (RAPTOR1B), in plants, we analyzed the effect of raptor1b mutations on growth and physiology in Arabidopsis (Arabidopsis thaliana) by detailed phenotyping, metabolomic, lipidomic, and proteomic analyses. Mutation of RAPTOR1B resulted in a strong reduction of TOR kinase activity, leading to massive changes in central carbon and nitrogen metabolism, accumulation of excess starch, and induction of autophagy. These shifts led to a significant reduction of plant growth that occurred nonlinearly during developmental stage transitions. This phenotype was accompanied by changes in cell morphology and tissue anatomy. In contrast to previous studies in rice (Oryza sativa), we found that the Arabidopsis raptor1b mutation did not affect chloroplast development or photosynthetic electron transport efficiency; however, it resulted in decreased CO2 assimilation rate and increased stomatal conductance. The raptor1b mutants also had reduced abscisic acid levels. Surprisingly, abscisic acid feeding experiments resulted in partial complementation of the growth phenotypes, indicating the tight interaction between TOR function and hormone synthesis and signaling in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Regulación de la Expresión Génica de las Plantas , Lípidos/química , Lípidos/genética , Meristema/genética , Meristema/fisiología , Mutación , Fijación del Nitrógeno/genética , Fotosíntesis/fisiología , Hojas de la Planta/anatomía & histología , Hojas de la Planta/fisiología , Hojas de la Planta/ultraestructura , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Especies Reactivas de Oxígeno/metabolismo , Metabolismo Secundario/genéticaRESUMEN
Hybrids often differ in fitness from their parents. They may be superior, translating into hybrid vigour or heterosis, but they may also be markedly inferior, because of hybrid weakness or incompatibility. The underlying genetic causes for the latter can often be traced back to genes that evolve rapidly because of sexual or host-pathogen conflicts. Hybrid weakness may manifest itself only in later generations, in a phenomenon called hybrid breakdown. We have characterized a case of hybrid breakdown among two Arabidopsis thaliana accessions, Shahdara (Sha, Tajikistan) and Lövvik-5 (Lov-5, Northern Sweden). In addition to chlorosis, a fraction of the F2 plants have defects in leaf and embryo development, and reduced photosynthetic efficiency. Hybrid chlorosis is due to two major-effect loci, of which one, originating from Lov-5, appears to encode an RNA helicase (AtRH18). To examine the role of the chlorosis allele in the Lövvik area, in addition to eight accessions collected in 2009, we collected another 240 accessions from 15 collections sites, including Lövvik, from Northern Sweden in 2015. Genotyping revealed that Lövvik collection site is separated from the rest. Crosses between 109 accessions from this area and Sha revealed 85 cases of hybrid chlorosis, indicating that the chlorosis-causing allele is common in this area. These results suggest that hybrid breakdown alleles not only occur at rapidly evolving loci, but also at genes that code for conserved processes.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes Recesivos , ARN Helicasas/genética , Alelos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Quimera , Clorofila/genética , Clorofila/metabolismo , Cromosomas de las Plantas , Regulación de la Expresión Génica de las Plantas , Vigor Híbrido , Fotosíntesis/genética , SueciaRESUMEN
The process of starch granule formation in leaves of Arabidopsis (Arabidopsis thaliana) is obscure. Besides STARCH SYNTHASE4 (SS4), the PLASTIDIAL PHOSPHORYLASE (PHS1) also seems to be involved, since dpe2-1/phs1a double mutants lacking both PHS1 and the cytosolic DISPROPORTIONATING ENZYME2 (DPE2) displayed only one starch granule per chloroplast under normal growth conditions. For further studies, a dpe2-1/phs1a/ss4 triple mutant and various combinations of double mutants were generated and metabolically analyzed with a focus on starch metabolism. The dpe2-1/phs1a/ss4 mutant revealed a massive starch excess phenotype. Furthermore, these plants grown under 12 h of light/12 h of dark harbored a single large and spherical starch granule per plastid. The number of starch granules was constant when the light/dark regime was altered, but this was not observed in the parental lines. With regard to growth, photosynthetic parameters, and metabolic analyses, the triple mutant additionally displayed alterations in comparison with ss4 and dpe2-1/phs1a The results clearly illustrate that PHS1 and SS4 are differently involved in starch granule formation and do not act in series. However, SS4 appears to exert a stronger influence. In connection with the characterized double mutants, we discuss the generation of starch granules and the observed formation of spherical starch granules.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastidios/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Almidón Sintasa/genética , Almidón Sintasa/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Sistema de la Enzima Desramificadora del Glucógeno/genética , Sistema de la Enzima Desramificadora del Glucógeno/metabolismo , Luz , Microscopía Electrónica , Mutación , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/efectos de la radiación , Proteínas Tirosina Fosfatasas/genética , Almidón/ultraestructuraRESUMEN
PsaI is the only subunit of PSI whose precise physiological function has not yet been elucidated in higher plants. While PsaI is involved in PSI trimerization in cyanobacteria, trimerization was lost during the evolution of the eukaryotic PSI, and the entire PsaI side of PSI underwent major structural remodelling to allow for binding of light harvesting complex II antenna proteins during state transitions. Here, we have generated a tobacco (Nicotiana tabacum) knockout mutant of the plastid-encoded psaI gene. We show that PsaI is not required for the redox reactions of PSI. Neither plastocyanin oxidation nor the processes at the PSI acceptor side are impaired in the mutant, and both linear and cyclic electron flux rates are unaltered. The PSI antenna cross section is unaffected, state transitions function normally, and binding of other PSI subunits to the reaction centre is not compromised. Under a wide range of growth conditions, the mutants are phenotypically and physiologically indistinguishable from wild-type tobacco. However, in response to high-light and chilling stress, and especially during leaf senescence, PSI content is reduced in the mutants, indicating that the I-subunit plays a role in stabilizing PSI complexes.