RESUMEN
The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.
Asunto(s)
Cloroplastos , Nicotiana , Synechococcus , Cloroplastos/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Synechococcus/metabolismo , Synechococcus/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Arabidopsis/metabolismo , Arabidopsis/genética , Bicarbonatos/metabolismo , Fotosíntesis , Proteínas de Transporte de Anión/metabolismo , Proteínas de Transporte de Anión/genética , Dióxido de Carbono/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
The evolution of photosynthesis and its associated metabolic pathways has been crucial to the successful establishment of plants, but has also challenged plant cells in the form of production of reactive oxygen species (ROS). Intriguingly, multiple forms of ROS are generated in virtually every plant cell compartment through diverse pathways. As a result, a sophisticated network of ROS detoxification and signaling that is simultaneously tailored to individual organelles and safeguards the entire cell is necessary. Here we take an organelle-centric view on the principal sources and sinks of ROS across the plant cell and provide insights into the ROS-induced organelle to nucleus retrograde signaling pathways needed for operational readjustments during environmental stresses.
Asunto(s)
Células Vegetales , Transducción de Señal , Núcleo Celular/metabolismo , Fotosíntesis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Signaling events triggered by hydrogen peroxide (H2O2) regulate plant growth and defense by orchestrating a genome-wide transcriptional reprogramming. However, the specific mechanisms that govern H2O2-dependent gene expression are still poorly understood. Here, we identify the Arabidopsis Mediator complex subunit MED8 as a regulator of H2O2 responses. The introduction of the med8 mutation in a constitutive oxidative stress genetic background (catalase-deficient, cat2) was associated with enhanced activation of the salicylic acid pathway and accelerated cell death. Interestingly, med8 seedlings were more tolerant to oxidative stress generated by the herbicide methyl viologen (MV) and exhibited transcriptional hyperactivation of defense signaling, in particular salicylic acid- and jasmonic acid-related pathways. The med8-triggered tolerance to MV was manipulated by the introduction of secondary mutations in salicylic acid and jasmonic acid pathways. In addition, analysis of the Mediator interactome revealed interactions with components involved in mRNA processing and microRNA biogenesis, hence expanding the role of Mediator beyond transcription. Notably, MED8 interacted with the transcriptional regulator NEGATIVE ON TATA-LESS, NOT2, to control the expression of H2O2-inducible genes and stress responses. Our work establishes MED8 as a component regulating oxidative stress responses and demonstrates that it acts as a negative regulator of H2O2-driven activation of defense gene expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Herbicidas/farmacología , Complejo Mediador/metabolismo , Estrés Oxidativo/fisiología , Amitrol (Herbicida)/farmacología , Arabidopsis/efectos de los fármacos , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Complejo Mediador/genética , MicroARNs , Estrés Oxidativo/efectos de los fármacos , Paraquat/farmacología , Plantas Modificadas Genéticamente , Dominios Proteicos , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/metabolismo , Factores Generales de Transcripción/genética , Factores Generales de Transcripción/metabolismoRESUMEN
The sulfur metabolism pathway in plants produces a variety of compounds that are central to the acclimation response to oxidative stresses such as drought and high light. Primary sulfur assimilation provides the amino acid cysteine, which is utilized in protein synthesis and as a precursor for the cellular redox buffer glutathione. In contrast, the secondary sulfur metabolism pathway produces sulfated compounds such as glucosinolates and sulfated peptides, as well as a corresponding by-product 3'-phosphoadenosine 5'-phosphate (PAP). Emerging evidence over the past decade has shown that secondary sulfur metabolism also has a crucial engagement during oxidative stress. This occurs across various cellular, tissue, and organismal levels including chloroplast-to-nucleus retrograde signalling events mediated by PAP, modulation of hormonal signalling by sulfated compounds and PAP, control of physiological responses such as stomatal closure, and potential regulation of plant growth. In this review, we examine the contribution of the different components of plant secondary metabolism to oxidative stress homeostasis, and how this pathway is metabolically regulated. We further outline the key outstanding questions in the field that are necessary to understand how and why this 'specialized' metabolic pathway plays significant roles in plant oxidative stress tolerance.
Asunto(s)
Arabidopsis/metabolismo , Estrés Oxidativo , Azufre/metabolismo , Arabidopsis/genética , Sequías , Regulación de la Expresión Génica de las Plantas , Metabolismo Secundario , Transducción de SeñalRESUMEN
Chloroplast retrograde signaling networks are vital for chloroplast biogenesis, operation, and signaling, including excess light and drought stress signaling. To date, retrograde signaling has been considered in the context of land plant adaptation, but not regarding the origin and evolution of signaling cascades linking chloroplast function to stomatal regulation. We show that key elements of the chloroplast retrograde signaling process, the nucleotide phosphatase (SAL1) and 3'-phosphoadenosine-5'-phosphate (PAP) metabolism, evolved in streptophyte algae-the algal ancestors of land plants. We discover an early evolution of SAL1-PAP chloroplast retrograde signaling in stomatal regulation based on conserved gene and protein structure, function, and enzyme activity and transit peptides of SAL1s in species including flowering plants, the fern Ceratopteris richardii, and the moss Physcomitrella patens Moreover, we demonstrate that PAP regulates stomatal closure via secondary messengers and ion transport in guard cells of these diverse lineages. The origin of stomata facilitated gas exchange in the earliest land plants. Our findings suggest that the conquest of land by plants was enabled by rapid response to drought stress through the deployment of an ancestral SAL1-PAP signaling pathway, intersecting with the core abscisic acid signaling in stomatal guard cells.
Asunto(s)
Adaptación Fisiológica , Evolución Biológica , Cloroplastos/metabolismo , Transducción de Señal , Viridiplantae/fisiología , Adenosina Difosfato , Embryophyta/fisiología , Peróxido de Hidrógeno/metabolismo , Transporte Iónico , Movimiento , Óxido Nítrico/metabolismo , Filogenia , Estomas de Plantas/fisiologíaRESUMEN
Homeostasis of metabolism and regulation of stress-signaling pathways are important for plant growth. The metabolite 3'-phosphoadenosine-5'-phosphate (PAP) plays dual roles as a chloroplast retrograde signal during drought and high light stress, as well as a toxic by-product of secondary sulfur metabolism, and thus, its levels are regulated by the chloroplastic phosphatase, SAL1. Constitutive PAP accumulation in sal1 mutants improves drought tolerance but can impair growth and alter rosette morphology. Therefore, it is of interest to derive strategies to enable controlled and targeted PAP manipulation that could enhance drought tolerance while minimizing the negative effects on plant growth. We systematically tested the potential and efficiency of multiple established transgenic manipulation tools in altering PAP levels in Arabidopsis. Dexamethasone (dex)-inducible silencing of SAL1 via hpRNAi [pOpOff:SAL1hpRNAi] yielded reduction in SAL1 transcript and protein levels, yet failed to significantly induce PAP accumulation. Surprisingly, this was not due to insufficient silencing of the inducible system, as constitutive silencing using a strong promoter to drive hpRNAi and amiRNA targeting the SAL1 transcript also failed to increase PAP content or induce a sal1-like plant morphology despite significantly reducing the SAL1 transcript levels. In contrast, using dex-inducible expression of SAL1 cDNA to complement an Arabidopsis sal1 mutant successfully modulated PAP levels and restored rosette growth in a dosage-dependent manner. Results from this inducible complementation system indicate that plants with intermediate PAP levels could have improved rosette growth without compromising its drought tolerance. Additionally, preliminary evidence suggests that SAL1 cDNA driven by promoters of genes expressed specifically during early developmental stages such as ABA-Insensitive 3 (ABI3) could be another potential strategy for studying and optimizing PAP levels and drought tolerance while alleviating the negative impact of PAP on plant growth in sal1. Thus, we have identified ways that can allow future dissection into multiple aspects of stress and developmental regulation mediated by this chloroplast signal.
RESUMEN
Organelle-nuclear retrograde signaling regulates gene expression, but its roles in specialized cells and integration with hormonal signaling remain enigmatic. Here we show that the SAL1-PAP (3'-phosphoadenosine 5'- phosphate) retrograde pathway interacts with abscisic acid (ABA) signaling to regulate stomatal closure and seed germination in Arabidopsis. Genetically or exogenously manipulating PAP bypasses the canonical signaling components ABA Insensitive 1 (ABI1) and Open Stomata 1 (OST1); priming an alternative pathway that restores ABA-responsive gene expression, ROS bursts, ion channel function, stomatal closure and drought tolerance in ost1-2. PAP also inhibits wild type and abi1-1 seed germination by enhancing ABA sensitivity. PAP-XRN signaling interacts with ABA, ROS and Ca2+; up-regulating multiple ABA signaling components, including lowly-expressed Calcium Dependent Protein Kinases (CDPKs) capable of activating the anion channel SLAC1. Thus, PAP exhibits many secondary messenger attributes and exemplifies how retrograde signals can have broader roles in hormone signaling, allowing chloroplasts to fine-tune physiological responses.
Asunto(s)
Ácido Abscísico/metabolismo , Adenosina Difosfato/metabolismo , Arabidopsis/fisiología , Cloroplastos/metabolismo , Germinación , Estomas de Plantas/fisiología , Transducción de Señal , Regulación de la Expresión Génica de las Plantas , Reguladores del Crecimiento de las Plantas/metabolismoRESUMEN
Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3'-phosphoadenosine 5'-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cloroplastos/metabolismo , Estrés Oxidativo , Monoéster Fosfórico Hidrolasas/metabolismo , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Disulfuros/metabolismo , Activación Enzimática , Regulación de la Expresión Génica de las Plantas , Glutatión , Oxidación-Reducción , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Multimerización de Proteína , Homología de Secuencia de Aminoácido , Transducción de Señal , Especificidad por SustratoRESUMEN
Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3'-phosphoadenosine 5'-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5'-3' exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response.
Asunto(s)
Adenosina Difosfato/metabolismo , Apoptosis/fisiología , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Transducción de Señal/fisiología , Apoptosis/genética , Arabidopsis/genética , Arabidopsis/microbiología , Clorofila/metabolismo , Cloroplastos/genética , Resistencia a la Enfermedad/genética , Mutación , Mio-Inositol-1-Fosfato Sintasa/genética , Mio-Inositol-1-Fosfato Sintasa/metabolismo , Oxidación-Reducción , Fotosíntesis/genética , Fotosíntesis/fisiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Inmunidad de la Planta/genética , Pseudomonas syringae/fisiología , Transducción de Señal/genéticaRESUMEN
The chloroplast can act as an environmental sensor, communicating with the cell during biogenesis and operation to change the expression of thousands of proteins. This process, termed retrograde signaling, regulates expression in response to developmental cues and stresses that affect photosynthesis and yield. Recent advances have identified many signals and pathways-including carotenoid derivatives, isoprenes, phosphoadenosines, tetrapyrroles, and heme, together with reactive oxygen species and proteins-that build a communication network to regulate gene expression, RNA turnover, and splicing. However, retrograde signaling pathways have been viewed largely as a means of bilateral communication between organelles and nuclei, ignoring their potential to interact with hormone signaling and the cell as a whole to regulate plant form and function. Here, we discuss new findings on the processes by which organelle communication is initiated, transmitted, and perceived, not only to regulate chloroplastic processes but also to intersect with cellular signaling and alter physiological responses.
Asunto(s)
Cloroplastos/fisiología , Regulación de la Expresión Génica de las Plantas , Fotosíntesis , Desarrollo de la Planta , Plantas/metabolismo , Cloroplastos/metabolismo , Plantas/genética , Transducción de SeñalRESUMEN
A key plant response to drought is the accumulation of specific sets of metabolites that act as osmoprotectants, osmolytes, antioxidants, and/or stress signals. An emerging question is: how do plants regulate metabolism to balance the 'competing interests' between metabolites during stress? Recent research connects primary sulfur metabolism (e.g., sulfate transport in the vasculature, its assimilation in leaves, and the recycling of sulfur-containing compounds) with the drought stress response. In this review, we highlight key steps in sulfur metabolism that play significant roles in drought stress signaling and responses. We propose that a complex balancing act is required to coordinate primary and secondary sulfur metabolism during the drought stress response in plants.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/fisiología , Fenómenos Fisiológicos de las Plantas , Plantas/metabolismo , Estrés Fisiológico/fisiología , Azufre/metabolismo , Transporte Biológico , Sequías , Estrés Oxidativo , Transducción de SeñalRESUMEN
Plant organelles produce retrograde signals to alter nuclear gene expression in order to coordinate their biogenesis, maintain homeostasis, or optimize their performance under adverse conditions. Many signals of different chemical nature have been described in the past decades, including chlorophyll intermediates, reactive oxygen species (ROS), and adenosine derivatives. While the effects of retrograde signaling on gene expression are well understood, the initiation and transport of the signals and their mode of action have either not been resolved, or are a matter of speculation. Moreover, retrograde signaling should be considered as part of a broader cellular network, instead of as separate pathways, required to adjust to changing physiologically relevant conditions. Here we summarize current plastid retrograde signaling models in plants, with a focus on new signaling pathways, SAL1-PAP, methylerythritol cyclodiphosphate (MEcPP), and ß-cyclocitral (ß-CC), and outline missing links or future areas of research that we believe need to be addressed to have a better understanding of plant intracellular signaling networks.