ABSTRACT
24 h cold exposure (4°C) is sufficient to reduce pathogen susceptibility in Arabidopsis thaliana against the virulent Pseudomonas syringae pv. tomato (Pst) strain even when the infection occurs five days later. This priming effect is independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) and can be observed in the immune-compromised eds1-2 null mutant. In contrast, cold priming-reduced Pst susceptibility is strongly impaired in knock-out lines of the stromal and thylakoid ascorbate peroxidases (sAPX/tAPX) highlighting their relevance for abiotic stress-related increased immune resilience. Here, we extended our analysis by generating an eds1 sapx double mutant. eds1 sapx showed eds1-like resistance and susceptibility phenotypes against Pst strains containing the effectors avrRPM1 and avrRPS4. In comparison to eds1-2, susceptibility against the wildtype Pst strain was constitutively enhanced in eds1 sapx. Although a prior cold priming exposure resulted in reduced Pst titers in eds1-2, it did not alter Pst resistance in eds1 sapx. This demonstrates that the genetic sAPX requirement for cold priming of basal plant immunity applies also to an eds1 null mutant background.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Ascorbate Peroxidases/metabolism , Gene Expression Regulation, Plant/genetics , Plant Diseases/genetics , Plant Immunity , Pseudomonas syringae , Thylakoids/metabolismABSTRACT
Exposure of Arabidopsis (Arabidopsis thaliana) to 4°C imprints a cold memory that modulates gene expression in response to a second (triggering) stress stimulus applied several days later. Comparison of plastid transcriptomes of cold-primed and control plants directly before they were exposed to the triggering stimulus showed downregulation of several subunits of chloroplast NADPH dehydrogenase (NDH) and regulatory subunits of ATP synthase. NDH is, like proton gradient 5 (PGR5)-PGR5-like1 (PGRL1), a thylakoid-embedded, ferredoxin-dependent plastoquinone reductase that protects photosystem I and stabilizes ATP synthesis by cyclic electron transport (CET). Like PGRL1A and PGRL1B transcript levels, ndhA and ndhD transcript levels decreased during the 24-h long priming cold treatment. PGRL1 transcript levels were quickly reset in the postcold phase, but expression of ndhA remained low. The transcript abundances of other ndh genes decreased within the next days. Comparison of thylakoid-bound ascorbate peroxidase (tAPX)-free and transiently tAPX-overexpressing or tAPX-downregulating Arabidopsis lines demonstrated that ndh expression is suppressed by postcold induction of tAPX. Four days after cold priming, when tAPX protein accumulation was maximal, NDH activity was almost fully lost. Lack of the NdhH-folding chaperonin Crr27 (Cpn60ß4), but not lack of the NDH activity modulating subunits NdhM, NdhO, or photosynthetic NDH subcomplex B2 (PnsB2), strengthened priming regulation of zinc finger of A. thaliana 10, which is a nuclear-localized target gene of the tAPX-dependent cold-priming pathway. We conclude that cold-priming modifies chloroplast-to-nucleus stress signaling by tAPX-mediated suppression of NDH-dependent CET and that plastid-encoded NdhH, which controls subcomplex A assembly, is of special importance for memory stabilization.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Photosynthetic Reaction Center Complex Proteins , Arabidopsis/genetics , Arabidopsis/metabolism , Ascorbate Peroxidases/genetics , Ascorbate Peroxidases/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , NADPH Dehydrogenase/genetics , NADPH Dehydrogenase/metabolism , Chloroplasts/metabolism , Photosystem I Protein Complex/genetics , Photosystem I Protein Complex/metabolism , Electron Transport , Adenosine Triphosphate/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Membrane Proteins/metabolismABSTRACT
Chloroplasts serve as cold priming hubs modulating the transcriptional response of Arabidopsis thaliana to a second cold stimulus for several days by postcold accumulation of thylakoid ascorbate peroxidases (tAPX). In an attempt to investigate cross-priming effects of cold on plant pathogen protection, we show here that such a single 24-h cold treatment at 4°C decreased the susceptibility of Arabidopsis to virulent Pseudomonas syringae pv. tomato DC3000 but did not alter resistance against the avirulent P. syringae pv. tomato avRPM1 and P. syringae pv. tomato avrRPS4 strains or the effector-deficient P. syringae pv. tomato strain hrcC-. The effect of cold priming against P. syringae pv. tomato was active immediately after cold exposure and memorized for at least 5 days. The priming benefit was established independent of the immune regulator Enhanced Disease Susceptibility 1 (EDS1) or activation of the immune-related genes NHL10, FRK1, ICS1 and PR1 but required thylakoid-bound as well as stromal ascorbate peroxidase activities because the effect was absent or weak in corresponding knock-out-lines. Suppression of tAPX postcold regulation in a conditional-inducible tAPX-RNAi line led to increased bacterial growth numbers. This highlights that the plant immune system benefits from postcold regeneration of the protective chloroplast peroxidase system.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Cold Temperature , Plant Diseases , Arabidopsis/enzymology , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Ascorbate Peroxidases/genetics , Ascorbate Peroxidases/metabolism , Disease Resistance , Gene Expression Regulation, Plant , Peroxidases/genetics , Peroxidases/metabolism , Plant Diseases/microbiology , Plastids/enzymology , Plastids/genetics , Pseudomonas syringae/pathogenicityABSTRACT
Cold-priming uncouples cold and light regulation of otherwise tightly co-regulated genes. In this study, we focused on the early regulatory processes in Arabidopsis within the first 2 h in cold and in high light after a 5-d lag-phase at 20 °C and 24 h cold-priming at 4 °C. Priming quickly modified gene expression in a trigger-specific manner. In the early stress-response phase during cold and high-light triggering, it reduced the regulatory amplitudes of many up- and down-regulated genes. A third of the priming-regulated genes were jasmonate-sensitive, including the full set of genes required for oxylipin biosynthesis. Analysis of wild-type and mutant plants based on qPCR demonstrated that biosynthesis of the jasmonic acid (JA) precursor 12-oxo phytenoic acid (OPDA) relative to the availability of JA dampened the response of the genes for oxylipin biosynthesis. In oxylipin biosynthetic mutants, cold-priming more strongly affected genes involved in the biosynthesis of OPDA than in its conversion to JA. In addition, priming-dependent dampening of the triggering response was more linked to OPDA than to regulation of the JA concentration. Spray application of OPDA prior to triggering counteracted the priming effect. Regulation of the oxylipin hub was controlled by modulation of the oxylipin-sensitivity of the genes for OPDA biosynthesis, but it was insensitive to priming-induced accumulation of thylakoid ascorbate peroxidase, thus identifying a parallel-acting cold-priming pathway.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclopentanes , Fatty Acids, Unsaturated , Gene Expression Regulation, Plant , OxylipinsABSTRACT
Cold slows down Calvin cycle activity stronger than photosynthetic electron transport, which supports production of reactive oxygen species (ROS). Even under extreme temperature conditions, most ROS are detoxified by the combined action of low-molecular weight antioxidants and antioxidant enzymes. Subsequent regeneration of the low-molecular weight antioxidants by NAD(P)H and thioredoxin/thiol-dependent pathways relaxes the electron pressure in the photosynthetic electron transport chain. In general, the chloroplast antioxidant system protects plants from severe damage of enzymes, metabolites, and cellular structures by both ROS detoxification and antioxidant recycling. Various methods have been developed to quantify ROS and antioxidant levels in photosynthetic tissues. Here, we summarize a series of exceptionally fast and easily applicable methods that show local ROS accumulation and provide information on the overall availability of reducing sugars, mainly ascorbate, and of thiols.
Subject(s)
Acclimatization , Antioxidants/metabolism , Cold Temperature , Plant Leaves/metabolism , Plant Physiological Phenomena , Reactive Oxygen Species/metabolism , Oxidation-Reduction , PhenotypeABSTRACT
BACKGROUND: The majority of stress-sensitive genes responds to cold and high light in the same direction, if plants face the stresses for the first time. As shown recently for a small selection of genes of the core environmental stress response cluster, pre-treatment of Arabidopsis thaliana with a 24 h long 4 °C cold stimulus modifies cold regulation of gene expression for up to a week at 20 °C, although the primary cold effects are reverted within the first 24 h. Such memory-based regulation is called priming. Here, we analyse the effect of 24 h cold priming on cold regulation of gene expression on a transcriptome-wide scale and investigate if and how cold priming affects light regulation of gene expression. RESULTS: Cold-priming affected cold and excess light regulation of a small subset of genes. In contrast to the strong gene co-regulation observed upon cold and light stress in non-primed plants, most priming-sensitive genes were regulated in a stressor-specific manner in cold-primed plant. Furthermore, almost as much genes were inversely regulated as co-regulated by a 24 h long 4 °C cold treatment and exposure to heat-filtered high light (800 µmol quanta m- 2 s- 1). Gene ontology enrichment analysis revealed that cold priming preferentially supports expression of genes involved in the defence against plant pathogens upon cold triggering. The regulation took place on the cost of the expression of genes involved in growth regulation and transport. On the contrary, cold priming resulted in stronger expression of genes regulating metabolism and development and weaker expression of defence genes in response to high light triggering. qPCR with independently cultivated and treated replicates confirmed the trends observed in the RNASeq guide experiment. CONCLUSION: A 24 h long priming cold stimulus activates a several days lasting stress memory that controls cold and light regulation of gene expression and adjusts growth and defence regulation in a stressor-specific manner.
Subject(s)
Arabidopsis/physiology , Cold Temperature , Gene Expression/physiology , Light , Transcriptome/drug effects , Arabidopsis/genetics , Arabidopsis/radiation effects , Gene Expression/radiation effectsABSTRACT
24 h exposure to 4 °C primes Arabidopsis thaliana in the pre-bolting rosette stage for several days against full cold activation of the ROS responsive genes ZAT10 and BAP1 and causes stronger cold-induction of pleiotropically stress-regulated genes. Transient over-expression of thylakoid ascorbate peroxidase (tAPX) at 20 °C mimicked and tAPX transcript silencing antagonized cold-priming of ZAT10 expression. The tAPX effect could not be replaced by over-expression of stromal ascorbate peroxidase (sAPX) demonstrating that priming is specific to regulation of tAPX availability and, consequently, regulated locally at the thylakoid membrane. Arabidopsis acquired cold primability in the early rosette stage between 2 and 4 weeks. During further rosette development, primability was widely maintained in the oldest leaves. Later formed and later maturing leaves were not primable demonstrating that priming is stronger regulated with plant age than with leaf age. In 4-week-old plants, which were strongest primable, the memory was fully erasable and lost seven days after priming. In summary, we conclude that cold-priming of chloroplast-to-nucleus ROS signalling by transient post-stress induction of tAPX transcription is a strategy to modify cell signalling for some time without affecting the alertness for activation of cold acclimation responses.
Subject(s)
Chloroplasts/metabolism , Reactive Oxygen Species/metabolism , Thylakoids/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Ascorbate Peroxidases/metabolism , Gene Expression Regulation, Plant/physiology , Peroxidases/metabolism , Plant Leaves/metabolism , Signal Transduction/physiologyABSTRACT
Cold is a major stressor, which limits plant growth and development in many parts of the world, especially in the temperate climate zones. A large number of experimental studies has demonstrated that not only acclimation and entrainment but also the experience of single short stress events of various abiotic or biotic kinds (priming stress) can improve the tolerance of plants to chilling temperatures. This process, called priming, depends on a stress "memory". It does not change cold sensitivity per se but beneficially modifies the response to cold and can last for days, months, or even longer. Elicitor factors and antagonists accumulate due to increased biosynthesis or decreased degradation either during or after the priming stimulus. Comparison of priming studies investigating improved tolerance to chilling temperatures highlighted key regulatory functions of ROS/RNS and antioxidant enzymes, plant hormones, especially jasmonates, salicylates, and abscisic acid, and signalling metabolites, such as ß- and γ-aminobutyric acid (BABA and GABA) and melatonin. We conclude that these elicitors and antagonists modify local and systemic cold tolerance by integration into cold-induced signalling cascades.
Subject(s)
Adaptation, Physiological/physiology , Cold-Shock Response/physiology , Plant Physiological Phenomena , Cold TemperatureABSTRACT
The redox imbalanced 6 mutant (rimb6) of Arabidopsis thaliana was isolated in a genetic screening approach for mutants with defects in chloroplast-to-nucleus redox signaling. It has an atypically low activation status of the 2-Cys peroxiredoxin-A promoter in the seedling stage. rimb6 shows wildtype-like germination, seedling development and greening, but slower growth and reduced biomass in the rosette stage. Mapping of the casual mutation revealed that rimb6 carries a single nucleotide polymorphism in the gene encoding CONSTITUTIVE EXPRESSER OF PATHOGENESIS RELATED (PR) GENES 1, CPR1 (At4g12560), leading to a premature stop codon. CPR1 is known as a repressor of pathogen signaling and regulator of microtubule organization. Allelism of rimb6 and cpr1 revealed a function of CPR1 in chloroplast stress protection. Expression studies in pathogen signaling mutants demonstrated that CPR1-mediated activation of genes for photosynthesis and chloroplast antioxidant protection is, in contrast to activation of pathogen responses, regulated independently from PAD4-controlled salicylic acid (SA) accumulation. We conclude that the support of plastid function is a basic, SA-independent function of CPR1.
ABSTRACT
BACKGROUND: The Arabidopsis ERFIb / RAP2.4 transcription factor family consists of eight members with highly conserved DNA binding domains. Selected members have been characterized individually, but a systematic comparison is pending. The redox-sensitive transcription factor RAP2.4a mediates chloroplast-to-nucleus redox signaling and controls induction of the three most prominent chloroplast peroxidases, namely 2-Cys peroxiredoxin A (2CPA) and thylakoid- and stromal ascorbate peroxidase (tAPx and sAPx). To test the specificity and redundancy of RAP2.4 transcription factors in the regulation of genes for chloroplast peroxidases, we compared the DNA-binding sites of the transcription factors in tertiary structure models, analyzed transcription factor and target gene regulation by qRT-PCR in RAP2.4, 2-Cys peroxiredoxin and ascorbate peroxidase T-DNA insertion lines and RAP2.4 overexpressing lines of Arabidopsis thaliana and performed promoter binding studies. RESULTS: All RAP2.4 proteins bound the tAPx promoter, but only the four RAP2.4 proteins with identical DNA contact sites, namely RAP2.4a, RAP2.4b, RAP2.4d and RAP2.4h, interacted stably with the redox-sensitive part of the 2CPA promoter. Gene expression analysis in RAP2.4 knockout lines revealed that RAP2.4a is the only one supporting 2CPA and chloroplast APx expression. Rap2.4h binds to the same promoter region as Rap2.4a and antagonizes 2CPA expression. Like the other six RAP2.4 proteins, Rap2.4 h promotes APx mRNA accumulation. Chloroplast ROS signals induced RAP2.4b and RAP2.4d expression, but these two transcription factor genes are (in contrast to RAP2.4a) insensitive to low 2CP availability, and their expression decreased in APx knockout lines. RAP2.4e and RAP2.4f gradually responded to chloroplast APx availability and activated specifically APx expression. These transcription factors bound, like RAP2.4c and RAP2.4g, the tAPx promoter, but hardly the 2CPA promoter. CONCLUSIONS: The RAP2.4 transcription factors form an environmentally and developmentally regulated transcription factor network, in which the various members affect the expression intensity of the others. Within the transcription factor family, RAP2.4a has a unique function as a general transcriptional activator of chloroplast peroxidase activity. The other RAP2.4 proteins mediate the fine-control and adjust the relative availability of 2CPA, sAPx and tAPx.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Chloroplasts/genetics , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Plant , Peroxidases/genetics , Transcription Factors/metabolism , Arabidopsis/enzymology , Arabidopsis Proteins/genetics , Ascorbate Peroxidases/genetics , Binding Sites , Chloroplasts/enzymology , DNA/metabolism , DNA, Bacterial , Gene Regulatory Networks , Peroxiredoxins/genetics , Promoter Regions, Genetic , Protein Binding , Reactive Oxygen Species/metabolism , Two-Hybrid System TechniquesABSTRACT
Priming improves an organism's performance upon a future stress. To test whether cold priming supports protection in spring and how it is affected by cold acclimation, we compared seven Arabidopsis accessions with different cold acclimation potentials in the field and in the greenhouse for growth, photosynthetic performance and reproductive fitness in March and May after a 14 day long cold-pretreatment at 4 °C. In the plants transferred to the field in May, the effect of the cold pretreatment on the seed yield correlated with the cold acclimation potential of the accessions. In the March transferred plants, the reproductive fitness was most supported by the cold pretreatment in the accessions with the weakest cold acclimation potential. The fitness effect was linked to long-term effects of the cold pretreatment on photosystem II activity stabilization and leaf blade expansion. The study demonstrated that cold priming stronger impacts on plant fitness than cold acclimation in spring in accessions with intermediate and low cold acclimation potential.
Subject(s)
Acclimatization/physiology , Arabidopsis/growth & development , Cold Temperature , Photosystem II Protein Complex/metabolism , Plant Leaves/growth & developmentABSTRACT
BACKGROUND: Short cold periods comprise a challenge to plant growth and development. Series of cold stresses improve plant performance upon a future cold stress. This effect could be provoked by priming, training or acclimation dependent hardening. Here, we compared the effect of 24 h (short priming stimulus) and of 2 week long cold-pretreatment (long priming stimulus) on the response of Arabidopsis thaliana to a single 24 h cold stimulus (triggering) after a 5 day long lag-phase, to test Arabidopsis for cold primability. RESULTS: Three types of pretreatment dependent responses were observed: (1) The CBF-regulon controlled gene COR15A was stronger activated only after long-term cold pretreatment. (2) The non-chloroplast specific stress markers PAL1 and CHS were more induced by cold after long-term and slightly stronger expressed after short-term cold priming. (3) The chloroplast ROS signaling marker genes ZAT10 and BAP1 were less activated by the triggering stimulus in primed plants. The effects on ZAT10 and BAP1 were more pronounced in 24 h cold-primed plants than in 14 day long cold-primed ones demonstrating independence of priming from induction and persistence of primary cold acclimation responses. Transcript and protein abundance analysis and studies in specific knock-out lines linked the priming-specific regulation of ZAT10 and BAP1 induction to the priming-induced long-term regulation of stromal and thylakoid-bound ascorbate peroxidase (sAPX and tAPX) expression. CONCLUSION: The plastid antioxidant system, especially, plastid ascorbate peroxidase regulation, transmits information on a previous cold stress over time without the requirement of establishing cold-acclimation. We hypothesize that the plastid antioxidant system serves as a priming hub and that priming-dependent regulation of chloroplast-to-nucleus ROS signaling is a strategy to prepare plants under unstable environmental conditions against unpredictable stresses by supporting extra-plastidic stress protection.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Ascorbate Peroxidases/metabolism , Gene Expression Regulation, Plant , Plastids/enzymology , Reactive Oxygen Species/metabolism , Thylakoid Membrane Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Ascorbate Peroxidases/genetics , Chloroplast Proteins/genetics , Chloroplast Proteins/metabolism , Chloroplasts/enzymology , Chloroplasts/genetics , Chloroplasts/metabolism , Cold Temperature , Oxidative Stress , Plastids/genetics , Plastids/metabolism , Thylakoid Membrane Proteins/genetics , Thylakoids/enzymology , Thylakoids/genetics , Thylakoids/metabolismABSTRACT
Temperature variations impact on the balance between photosynthetic electron transport and electron-consuming assimilation reactions and transiently increase generation of reactive oxygen species (ROS). Previous studies demonstrated that the expression of C-repeat binding factors (CBFs), which activate cold acclimation reactions, respond to chloroplast ROS signals and that cold deacclimation is partly halted for days after the transfer of acclimated plants to optimal growth conditions in four Arabidopsis accessions from cold-continental habitats. We hypothesized that these accessions differ from others in the regulation of the plastid antioxidant system (PAS). In the present study, we compared the expression intensity of the 12 most prominent PAS genes for peroxidases, superoxide dismutase and low molecular weight antioxidant regenerating enzymes in 10 Arabidopsis accessions with regulation of CBF and COR (cold regulated genes) transcript levels and cold-regulated metabolite levels prior to cold, after 2 week long cold acclimation and during the first 3 days of deacclimation. In the accessions with prolonged activation of cold responses, by trend, weaker induction of various cold-inducible PAS genes and stronger decreases in the expression of negatively cold-regulated PAS genes were observed. Low PAS gene expression delayed the post-cold decrease in H2O2 levels after transfer of the plants from cold to optimal growth conditions. We conclude that weaker expression of various PAS genes in the cold is an adapted strategy of the Arabidopsis accessions N14, N13, Ms-0, and Kas-1 to avoid full inactivation of cold-responses in the first days after the end of the cold period.
ABSTRACT
Experience and memory of environmental stimuli that indicate future stress can prepare (prime) organismic stress responses even in species lacking a nervous system. The process through which such organisms prepare their phenotype for an improved response to future stress has been termed 'priming'. However, other terms are also used for this phenomenon, especially when considering priming in different types of organisms and when referring to different stressors. Here we propose a conceptual framework for priming of stress responses in bacteria, fungi and plants which allows comparison of priming with other terms, e.g. adaptation, acclimation, induction, acquired resistance and cross protection. We address spatial and temporal aspects of priming and highlight current knowledge about the mechanisms necessary for information storage which range from epigenetic marks to the accumulation of (dormant) signalling molecules. Furthermore, we outline possible patterns of primed stress responses. Finally, we link the ability of organisms to become primed for stress responses (their 'primability') with evolutionary ecology aspects and discuss which properties of an organism and its environment may favour the evolution of priming of stress responses.
Subject(s)
Bacterial Physiological Phenomena , Fungi/physiology , Plant Physiological Phenomena , Stress, Physiological/physiology , Adaptation, Physiological , Biological Evolution , EcologyABSTRACT
During low temperature exposure, Arabidopsis thaliana and many other plants from temperate climates increase in freezing tolerance in a process termed cold acclimation. However, the correct timing and rate of deacclimation, resulting in loss of freezing tolerance and initiation of growth is equally important for plant fitness and survival. While the molecular basis of cold acclimation has been investigated in detail, much less information is available about deacclimation. We have characterized the responses of 10 natural accessions of Arabidopsis thaliana that vary widely in their freezing tolerance, to deacclimation conditions. Sugar, proline and transcript levels declined sharply over three days in all accessions after transfer of cold acclimated plants to ambient temperatures, while freezing tolerance only declined in tolerant accessions. Correlations between freezing tolerance and the expression levels of COR genes and the content of glucose, fructose and sucrose, as well as many correlations among transcript and solute levels, that were highly significant in cold acclimated plants, were lost during deacclimation. Other correlations persisted, indicating that after three days of deacclimation, plant metabolism had not completely reverted back to the non-acclimated state. These data provide the basis for further molecular and genetic studies to unravel the regulation of deacclimation.
Subject(s)
Acclimatization/physiology , Arabidopsis/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cold Temperature , Freezing , Fructose/analysis , Glucose/analysis , Plant Leaves/genetics , Plant Leaves/metabolism , Raffinose/analysisABSTRACT
The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system.
ABSTRACT
Reactive oxygen species (ROS) are produced in plants under both non-stressful and stressful conditions. Various histochemical staining methods have been developed and are widely used to visualize ROS accumulation sites. In contrast to qualitative analysis, quantification of ROS has been time- and labor consuming. As a consequence, the number of samples, which could be analyzed in parallel, has been limited. To overcome this problem, we introduce an improved semiquantitative method, in which ROS levels are quantified after histochemical staining in plant organs with the digital image analysis package ImageJ.
Subject(s)
Hydrogen Peroxide/analysis , Plant Leaves/cytology , Staining and Labeling/methods , Superoxides/analysis , Nitroblue Tetrazolium/chemistry , Photography , Time FactorsABSTRACT
All genes encoding chloroplast antioxidant enzymes are nuclear-encoded and posttranscriptionally targeted to chloroplasts. The transcript levels of most of them decreased upon sucrose feeding like the transcript levels of many genes encoding components of the photosynthetic electron transport chain. However, the transcript abundance of stromal ascorbate peroxidase (s-APX; At4g08390) increased. Due to mild sugar application conditions, the plants kept the phosphorylation status of the ADP+ATP pool and the redox states of the NADPH+NADP+ and the ascorbate pools under control, which excludes them as signals in s-APX regulation. Correlation with ascorbate pool size regulation and comparison of transcript abundance regulation in the starch-biosynthetic mutant adg1, the ascorbate biosynthesis mutant vtc1, and the abscisic acid (ABA) biosynthetic mutant aba2 showed a link between sugar induction of s-APX and ascorbate biosynthesis.
Subject(s)
Antioxidants/metabolism , Arabidopsis/cytology , Arabidopsis/metabolism , Ascorbate Peroxidases/genetics , Ascorbic Acid/biosynthesis , Carbohydrate Metabolism , Chloroplasts/metabolism , Abscisic Acid/biosynthesis , Anthocyanins/metabolism , Arabidopsis/drug effects , Arabidopsis/radiation effects , Carbohydrate Metabolism/drug effects , Carbohydrate Metabolism/radiation effects , Chloroplasts/drug effects , Chloroplasts/radiation effects , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/radiation effects , Light , Mutation , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Leaves/radiation effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Starch/biosynthesis , Sucrose/metabolism , Sucrose/pharmacologyABSTRACT
Two collections of Arabidopsis GAL4 enhancer trap lines were screened for light-intensity dependent reporter gene activation. Line N9313 was isolated for its strong light-intensity regulation. The T-DNA element trapped distant enhancers of the SIG5 promoter, which drives expression of a sigma factor involved in regulation of chloroplast genes for photosystem II core proteins. The T-DNA insertion 715 bp upstream of the transcription initiation site splits the promoter in a distal and proximal part. Both parts are sensitive to blue and red light and depend on photosynthetic electron transport activity between photosystem II and the plastoquinone pool. The mainblue-light sensitivity is localized within a 196-bp sequence (-887 to -691 bp) in the proximal promoter region It is preferentially CRY1 and PHYB controlled. Type-I and type-II phytochromes mediate red-light sensitivity via various promoter elements spread over the proximal and distal upstream region. This work characterizes SIG5 as an anterograde control factor of chloroplast gene expression, which is controlled by chloroplast signals in a retrograde manner.
ABSTRACT
miR398 links expression of the three major chloroplast copper proteins, plastocyanin, CCS1 and Csd2, to copper availability. miR398 abundance was stronger plastocyanin-controlled in accessions from cold and continental habitats (Kas-1, Ms-0, WS) than in Cvi-0 and Col-0. Target gene regulation was broken for Csd2 in Cvi-0 upon cold-treatment. Comparison of miR398 levels, target gene regulation as well as Ago1 and miR168 expression demonstrated that the miR398 regulon can be overwritten by accession specific transcriptional regulation in Cvi-0. It is concluded that the escape from the miRNA control of copper homeostasis is linked to adaptation of Cvi-0 to its harsh natural habitat.