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During land plant evolution, the number of genes encoding for components of the thiol redox regulatory network and the generator systems of reactive oxygen species (ROS) expanded, tentatively indicating that they have a role in tailored environmental acclimatization. This hypothesis has been validated both experimentally and theoretically during the last few decades. Recent developments of dynamic redox-sensitive GFP (roGFP)-based in vivo sensors for H2O2 and the redox potential of the glutathione pool have paved the way for dissecting the kinetics changes that occur in these crucial parameters in response to environmental stressors. The versatile cellular redox sensory and response regulatory system monitors alterations in redox metabolism and controls the activity of redox target proteins, and thereby affects most, if not all, cellular processes ranging from transcription to translation and metabolism. This review uses examples to describe the role of the redox- and ROS-dependent regulatory network in realising the appropriate responses to diverse environmental stresses. The selected case studies concern different environmental challenges, namely excess excitation energy, the heavy metal cadmium and the metalloid arsenic, nitrogen or phosphate shortages as examples for nutrient deficiency, wounding, and nematode infestation. Each challenge affects the redox-regulatory and ROS network, but our present state of knowledge also points toward pressing questions that remain open in relation to the translation of redox regulation to environmental acclimatization., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Dark-light and light-dark transitions during the day are switching points of leaf metabolism that strongly affect the regulatory state of the cells, and this change is hypothesized to affect the translatome. The cytosolic glyceraldehyde-3-phosphate dehydrogenases GAPC1 and GAPC2 function in glycolysis, and carbohydrate and energy metabolism, but GAPC1/C2 also shows moonlighting functions in gene expression and post-transcriptional regulation. In this study we examined the rapid reprogramming of the translatome that occurs within 10 min at the end of the night and the end of the day in wild-type (WT) Arabidopsis and a gapc1/c2 double-knockdown mutant. Metabolite profiling compared to the WT showed that gapc1/c2 knockdown led to increases in a set of metabolites at the start of day, particularly intermediates of the citric acid cycle and linked pathways. Differences in metabolite changes were also detected at the end of the day. Only small sets of transcripts changed in the total RNA pool; however, RNA-sequencing revealed major alterations in polysome-associated transcripts at the light-transition points. The most pronounced difference between the WT and gapc1/c2 was seen in the reorganization of the translatome at the start of the night. Our results are in line with the proposed hypothesis that GAPC1/C2 play a role in the control of the translatome during light/dark transitions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: The goal of this study was to characterize biopolymers from two modes of the Herbaspirillum lusitanum P6-12 growth: planktonic, in which cells are free swimming, and biofilm life style, in which the cells are sessile., Methods and Results: Differences in biopolymers composition from planktonic and biofilm cells of H. lusitanum strain P6-12 were analysed using Fourier transform infrared spectroscopy (FTIR), sodium dodecyl sulphate-polyacrylamide gel electrophoresis, gas-liquid chromatography and spectrophotometry. A high degree of polymer separation and purification was achieved by ultracentrifugation, and column chromatography allowed us to identify the chemical differences between biopolymers from biofilm and planktonic H. lusitanum. It was shown that planktonic cells of H. lusitanum P6-12 when cultivated in a liquid medium to the end of the exponential phase of growth, produced two high-molecular-weight glycoconjugates (were arbitrarily called CPS-I and CPS-II) of a lipopolysaccharide (LPS) nature and a lipid-polysacharide complex (were arbitrarily called EPS). The EPS, CPS-I, CPS-II had different monosaccharide and lipid compositions. The extracellular polymeric matrix (EPM) produced by the biofilm cells was mostly proteinaceous, with a small amount of carbohydrates (up to 3%). From the biofilm culture medium, a free extracellular polymeric substance (was arbitrarily called fEPS) was obtained that contained proteins and carbohydrates (up to 7%). The cells outside the biofilm had capsules containing high-molecular-weight glycoconjugate (was arbitrarily called CPS FBC ) that consisted of carbohydrates (up to 10%), proteins (up to 16%) and lipids (up to 70%)., Conclusions: During biofilm formation, the bacteria secreted surface biopolymers that differed from those of the planktonic cells. The heterogeneity of the polysaccharide containing biopolymers of the H. lusitanum P6-12 surface is probably conditioned by their different functions in plant colonization and formation of an efficient symbiosis, as well as in cell adaptation to existence in plant tissues., Significance and Impact of the Study: The results of the study permit a better understanding of the physiological properties of the biopolymers, for example, in plant-microbe interactions., (© 2020 The Society for Applied Microbiology.)

Arabidopsis (Arabidopsis thaliana) 12-oxophytodienoic acid reductase isoform 3 (OPR3) is involved in the synthesis of jasmonic acid (JA) by reducing the α,β-unsaturated double bond of the cyclopentenone moiety in 12-oxophytodienoic acid (12-OPDA). Recent research revealed that JA synthesis is not strictly dependent on the peroxisomal OPR3. The ability of OPR3 to reduce trinitrotoluene suggests that the old yellow enzyme homolog OPR3 has additional functions. Here, we show that OPR3 catalyzes the reduction of a wide spectrum of electrophilic species that share a reactivity toward the major redox buffers glutathione (GSH) and ascorbate (ASC). Furthermore, we show that 12-OPDA reacts with ASC to form an ASC-12-OPDA adduct, but in addition OPR3 has the ability to regenerate ASC from monodehydroascorbate. The presented data characterize OPR3 as a bifunctional enzyme with NADPH-dependent α,β-ketoalkene double-bond reductase and monodehydroascorbate reductase activities (MDHAR). opr3 mutants showed a slightly less-reduced ASC pool in leaves in line with the MDHAR activity of OPR3 in vitro. These functions link redox homeostasis as mediated by ASC and GSH with OPR3 activity and metabolism of reactive electrophilic species., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Sulfur assimilation and sulfur metabolism are tightly controlled at the transcriptional, post-transcriptional, and post-translational levels in order to meet the demand for reduced sulfur in growth and metabolism. These regulatory mechanisms coordinate the cellular sulfhydryl supply with carbon and nitrogen assimilation in particular. Redox homeostasis is an important cellular parameter intimately connected to sulfur by means of multiple thiol modifications. Post-translational thiol modifications such as disulfide formation, sulfenylation, S-nitrosylation, persulfidation, and S-glutathionylation allow for versatile switching and adjustment of protein functions. This review focuses on redox-regulation of enzymes involved in the sulfur assimilation pathway, namely adenosine 5´-phosphosulfate reductase (APR), adenosine 5´-phosphosulfate kinase (APSK), and γ-glutamylcysteine ligase (GCL). The activity of these enzymes is adjusted at the transcriptional and post-translational level depending on physiological requirements and the state of the redox and reactive oxygen species network, which are tightly linked to abiotic stress conditions. Hormone-dependent fine-tuning contributes to regulation of sulfur assimilation. Thus, the link between oxylipin signalling and sulfur assimilation has been substantiated by identification of the so-called COPS module in the chloroplast with its components cyclophilin 20-3, O-acetylserine thiol lyase, 2-cysteine peroxiredoxin, and serine acetyl transferase. We now have a detailed understanding of how regulation enables the fine-tuning of sulfur assimilation under both normal and abiotic stress conditions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Forty years ago, 12-oxophytodienoic acid (12-OPDA) was reported as a prostaglandin (PG)-like metabolite of linolenic acid found in extracts of flaxseed. Since then, numerous studies have determined the role of 12-OPDA in regulating plant immunity, seed dormancy, and germination. This review summarizes our current knowledge of the regulation of 12-OPDA synthesis in the chloroplast and 12-OPDA-dependent signaling in gene expression and targeting protein functions. We describe the properties of OPDA that are linked to the activities of PGs, which are derived from arachidonic acid and act as tissue hormones in animals, including humans. The similarity of OPDA with bioactive PGs is particularly evident for the most-studied cyclopentenone, PG 15-dPGJ2. In addition to chemical approaches towards 12-OPDA synthesis, bio-organic synthesis strategies for 12-OPDA and analogous substances have recently been established. The resulting availability of OPDA will aid the identification of additional effector proteins, help in elucidating the mechanisms of OPDA sensing and transmission, and will foster the analysis of the physiological responses to OPDA in plants. There is a need to determine the compartmentation and transport of 12-OPDA and its conjugates, over long distances as well as short. It will be important to further study OPDA in animal and human cells, for example with respect to beneficial anti-inflammatory and anti-cancer activities.

Redox regulation, antioxidant defence, and reactive oxygen species (ROS) signalling are critical in performing and tuning metabolic activities. However, our concepts have mostly been developed for C3 plants since Arabidopsis thaliana has been the major model for research. Efforts to convert C3 plants to C4 to increase yield (such as IRRI's C4 Rice Project) entail a better understanding of these processes in C4 plants. Various photosynthetic enzymes that take part in light reactions and carbon reactions are regulated via redox components, such as thioredoxins as redox transmitters and peroxiredoxins. Hence, understanding redox regulation in the mesophyll and bundle sheath chloroplasts of C4 plants is of paramount importance: it appears impossible to utilize efficient C4 photosynthesis without understanding its exact redox needs and the regulation mechanisms used during light reactions. In this review, we discuss current knowledge on redox regulation in C3 and C4 plants, with special emphasis on the mesophyll and bundle sheath differences that are found in C4. In these two cell types in C4 plants, linear and cyclic electron transport in the chloroplasts operate differentially when compared to C3 chloroplasts, changing the redox needs of the cell. Therefore, our focus is on photosynthetic light reactions, ROS production dynamics, antioxidant defence, and thiol-based redox regulation, with the aim of providing an overview of our current knowledge.

Sugar beet is among the most salt-tolerant crops. This study aimed to investigate the metabolic adaptation of sugar beet to salt stress at the cellular and subcellular levels. Seedlings were grown hydroponically and subjected to stepwise increases in salt stress up to 300 mM NaCl. Highly enriched fractions of chloroplasts were obtained by non-aqueous fractionation using organic solvents. Total leaf metabolites and metabolites in chloroplasts were profiled at 3 h and 14 d after reaching the maximum salinity stress of 300 mM NaCl. Metabolite profiling by gas chromatography-mass spectrometry (GC-MS) resulted in the identification of a total of 83 metabolites in leaves and chloroplasts under control and stress conditions. There was a lower abundance of Calvin cycle metabolites under salinity whereas there was a higher abundance of oxidative pentose phosphate cycle metabolites such as 6-phosphogluconate. Accumulation of ribose-5-phosphate and ribulose-5-phosphate coincided with limitation of carbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Increases in glycolate and serine levels indicated that photorespiratory metabolism was stimulated in salt-stressed sugar beet. Compatible solutes such as proline, mannitol, and putrescine accumulated mostly outside the chloroplasts. Within the chloroplast, putrescine had the highest relative level and probably assisted in the acclimation of sugar beet to high salinity stress. The results provide new information on the contribution of chloroplasts and the extra-chloroplast space to salinity tolerance via metabolic adjustment in sugar beet., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Fine-tuned and coordinated regulation of transport, metabolism and redox homeostasis allows plants to acclimate to osmotic and ionic stress caused by high salinity. Sugar beet is a highly salt tolerant crop plant and is therefore an interesting model to study sodium chloride (NaCl) acclimation in crops. Sugar beet plants were subjected to a final level of 300 mM NaCl for up to 14 d in hydroponics. Plants acclimated to NaCl stress by maintaining its growth rate and adjusting its cellular redox and reactive oxygen species (ROS) network. In order to understand the unusual suppression of ROS accumulation under severe salinity, the regulation of elements of the redox and ROS network was investigated at the transcript level. First, the gene families of superoxide dismutase (SOD), peroxiredoxins (Prx), alternative oxidase (AOX), plastid terminal oxidase (PTOX) and NADPH oxidase (RBOH) were identified in the sugar beet genome. Salinity induced the accumulation of Cu-Zn-SOD, Mn-SOD, Fe-SOD3, all AOX isoforms, 2-Cys-PrxB, PrxQ, and PrxIIF. In contrast, Fe-SOD1, 1-Cys-Prx, PrxIIB and PrxIIE levels decreased in response to salinity. Most importantly, RBOH transcripts of all isoforms decreased. This pattern offers a straightforward explanation for the low ROS levels under salinity. Promoters of stress responsive antioxidant genes were analyzed in silico for the enrichment of cis-elements, in order to gain insights into gene regulation. The results indicate that special cis-elements in the promoters of the antioxidant genes in sugar beet participate in adjusting the redox and ROS network and are fundamental to high salinity tolerance of sugar beet., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

2-Cysteine peroxiredoxins (2-CysPrxs) switch between functions as a thiol peroxidase, chaperone, an interaction partner and possibly a proximity-based oxidase in a redox-dependent manner. In photosynthetic eukaryotes, 2-CysPrx localizes to the plastid, functions in the context of photosynthesis and enables an ascorbate peroxidase-independent water-water cycle for detoxifying H 2 O 2 The high degree of evolutionary conservation of 2-CysPrx suggests that the switching is an essential characteristic and needed to transduce redox information to downstream pathways and regulation. The study aimed at exploring the dissociation behavior of 2-CysPrx and its interactions with cyclophilin depending on bulk phase conditions. Isothermal titration microcalorimetry (ITC), dynamic light scattering and size exclusion chromatography (SEC) proved the previously suggested model that reduced 2-CysPrx below a critical transition concentration (CTC) exists in its dimeric state, and above the CTC adopts the decameric state. The presence of cyclophilin 20-3 (Cyp20-3) affected the CTC of a 2-CysPrx decamer suggesting interaction which was further quantified by direct titration of 2-CysPrx with Cyp20-3, and in overlays. Finally catalytic inactivation assays showed the higher catalytic efficiency of 2-CysPrx at pH 8 compared with pH 7.2, but also revealed increased inactivation by hyperoxidation at pH 8. Interestingly, calculation of the average turnover number until inactivation gave rather similar values of 243 and 268 catalytic cycles at pH 8 and pH 7.2, respectively. These quantitative data support a model where 2-CysPrx and Cyp20-3, by interaction, form a redox-sensitive regulatory module in the chloroplast which is under control of the photosynthesis-linked stromal pH value, the redox state and additional stromal protein factor(s)., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Silicon (Si) modulates tolerance to abiotic stresses, but little is known about the reversibility of stress effects by supplementing previously stressed plants with Si. This is surprising since recovery experiments might allow mechanisms of Si-mediated amelioration to be addressed. Rice was exposed to 10 µM CdCl2 for 4 d in hydroponics, followed by 0.6mM Si(OH)4 supplementation for 4 d. Si reversed the effects of Cd, as reflected in plant growth, photosynthesis, elemental composition, and some biochemical parameters. Cd-dependent deregulation of nutrient homeostasis was partially reversed by Si supply. Photosynthetic recovery within 48h following Si supply, coupled with strong stimulation of the ascorbate-glutathione system, indicates efficient activation of defense. The response was further verified by transcript analyses with emphasis on genes encoding members of the stress-associated protein (SAP) family. The transcriptional response to Cd was mostly reversed following Si supply. Reprogramming of the Cd response was obvious for Phytochelatin synthase 1, SAP1 , SAP14, and the transcription factor genes AP2/Erf020, Hsf31, and NAC6 whose transcript levels were strongly activated in roots of Cd-stressed rice, but down-regulated in the presence of Si. These findings, together with changes in biochemical parameters, highlight the significance of Si in growth recovery of Cd-stressed rice and indicate a decisive role for readjusting cell redox homeostasis., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

A diagnostic test system was developed to determine the toxicity of nanomaterials to the saltwater microalga Dunaliella salina through evaluation of cell death and changes in the culture growth rate at various toxicant concentrations, providing LC50 and other toxicological metrics. The viability of cells was shown to decrease with decreasing chlorophyll absorption of red light by damaged cells. This correlation was confirmed by independent fluorescence microscopic measurements of live and dead cells in the population. Two standard colorless pollutants, hydrogen peroxide and formaldehyde, were used to validate the colorimetric method. The method's performance is exemplified with three Ag-containing preparations (Ag nitrate, Ag proteinate, and 20-nm Ag nanoparticles) and with cetyltrimethylammonium bromide (CTAB) mixed with colloidal 15-nm Au and 20-nm Ag nanoparticles. The toxicity of the Ag-containing preparations to D. salina decreased in the order Ag nitrate ≥ Ag proteinate ≫ colloidal Ag. The toxicity of colloidal Au-CTAB mixtures was found to depend mostly on the content of free CTAB. The toxicity of colloidal Ag increased substantially in the presence of CTAB. The results suggest that our D. salina-based colorimetric test system can be used for simple and rapid preliminary screening of the toxicity of different nanomaterials., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Glutathione peroxidases (Gpxs) are antioxidant enzymes not studied so far in legume nodules, despite the fact that reactive oxygen species are produced at different steps of the symbiosis. The function of two Gpxs that are highly expressed in nodules of the model legume Lotus japonicus was examined. Gene expression analysis, enzymatic and nitrosylation assays, yeast cell complementation, in situ mRNA hybridization, immunoelectron microscopy, and LjGpx-green fluorescent protein (GFP) fusions were used to characterize the enzymes and to localize each transcript and isoform in nodules. The LjGpx1 and LjGpx3 genes encode thioredoxin-dependent phospholipid hydroperoxidases and are differentially regulated in response to nitric oxide (NO) and hormones. LjGpx1 and LjGpx3 are nitrosylated in vitro or in plants treated with S-nitrosoglutathione (GSNO). Consistent with the modification of the peroxidatic cysteine of LjGpx3, in vitro assays demonstrated that this modification results in enzyme inhibition. The enzymes are highly expressed in the infected zone, but the LjGpx3 mRNA is also detected in the cortex and vascular bundles. LjGpx1 is localized to the plastids and nuclei, and LjGpx3 to the cytosol and endoplasmic reticulum. Based on yeast complementation experiments, both enzymes protect against oxidative stress, salt stress, and membrane damage. It is concluded that both LjGpxs perform major antioxidative functions in nodules, preventing lipid peroxidation and other oxidative processes at different subcellular sites of vascular and infected cells. The enzymes are probably involved in hormone and NO signalling, and may be regulated through nitrosylation of the peroxidatic cysteine essential for catalytic function., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Like no other chemical or physical parameter, the natural light environment of plants changes with high speed and jumps of enormous intensity. To cope with this variability, photosynthetic organisms have evolved sensing and response mechanisms that allow efficient acclimation. Most signals originate from the chloroplast itself. In addition to very fast photochemical regulation, intensive molecular communication is realized within the photosynthesizing cell, optimizing the acclimation process. Current research has opened up new perspectives on plausible but mostly unexpected complexity in signalling events, crosstalk, and process adjustments. Within seconds and minutes, redox states, levels of reactive oxygen species, metabolites, and hormones change and transmit information to the cytosol, modifying metabolic activity, gene expression, translation activity, and alternative splicing events. Signalling pathways on an intermediate time scale of several minutes to a few hours pave the way for long-term acclimation. Thereby, a new steady state of the transcriptome, proteome, and metabolism is realized within rather short time periods irrespective of the previous acclimation history to shade or sun conditions. This review provides a time line of events during six hours in the 'stressful' life of a plant., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Regulation of the expression of nuclear genes encoding chloroplast proteins allows for metabolic adjustment in response to changing environmental conditions. This regulation is linked to retrograde signals that transmit information on the metabolic state of the chloroplast to the nucleus. Transcripts of several APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERF-TFs) were found to respond within 10 min after transfer of low-light-acclimated Arabidopsis thaliana plants to high light. Initiation of this transcriptional response was completed within 1 min after transfer to high light. The fast responses of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105, were entirely deregulated in triose phosphate/phosphate translocator (tpt) mutants. Similarly, activation of MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) was upregulated after 1 min in the wild type but not in the tpt mutant. Based on this, together with altered transcript regulation in mpk6 and erf6 mutants, a retrograde signal transmission model is proposed starting with metabolite export through the triose phosphate/phosphate translocator with subsequent MPK6 activation leading to initiation of AP2/ERF-TF gene expression and other downstream gene targets. The results show that operational retrograde signaling in response to high light involves a metabolite-linked pathway in addition to previously described redox and hormonal pathways.

2-Cysteine peroxiredoxins (2-CysPrxs) are ubiquitous and highly abundant proteins that serve multiple functions as peroxidases, chaperones, and thiol oxidases and in redox-dependent cell signalling. The chloroplast protein plays a role in seedling development and protection of the photosynthetic apparatus. This study aimed to unequivocally link conformation and function. To this end, a set of non-tagged site-directed mutagenized At2-CysPrx variants was engineered, which mimicked the conformational states and their specific functions: hyperoxidized form (C54D), reduced form (C54S, C176S), oxidized form (C54DC176K), phosphorylated form (T92D), reduced ability for oligomerization by interfering with the dimer-dimer interface (F84R) and a C-terminally truncated form [ΔC (-20 aa)]. These variants were fully or partly fixed in their quaternary structure and function, respectively, and were analysed for their conformational state and peroxidase and chaperone activity, as well as for their sensitivity to hyperoxidation. The presence of a His6-tag strongly influenced the properties of the protein. The ΔC variant became insensitive to hyperoxidation, while T92D and F84R became more sensitive. The C54D variant revealed the highest chaperone activity. The highest peroxidase activity was observed for the F84R and ΔC variants. Efficient interaction with NADP-dependent thioredoxin reductase C depended on the presence of Cys residues and the C-terminal tail. The results suggest that the structural flexibility is important for the switch between peroxidase and chaperone function and that evolution has conserved the functional switch instead of maximizing a single function. These variants are ideal tools for future conformation-specific studies in vivo and in vitro.

Chloroplasts are equipped with a nuclear-encoded antioxidant defence system the components of which are usually expressed at high transcript and activity levels. To significantly challenge the chloroplast antioxidant system, Arabidopsis thaliana plants, acclimated to extremely low light slightly above the light compensation point or to normal growth chamber light, were moved to high light corresponding to a 100- and 10-fold light jump, for 6 h and 24 h in order to observe the responses of the water-water cycle at the transcript, protein, enzyme activity, and metabolite levels. The plants coped efficiently with the high light regime and the photoinhibition was fully reversible. Reactive oxygen species (ROS), glutathione and ascorbate levels as well as redox states, respectively, revealed no particular oxidative stress in low-light-acclimated plants transferred to 100-fold excess light. Strong regulation of the water-water cycle enzymes at the transcript level was only partly reflected at the protein and activity levels. In general, low light plants had higher stromal (sAPX) and thylakoid ascorbate peroxidase (tAPX), dehydroascorbate reductase (DHAR), and CuZn superoxide dismutase (CuZnSOD) protein contents than normal light-grown plants. Mutants defective in components relevant for retrograde signalling, namely stn7, ex1, tpt1, and a mutant expressing E .coli catalase in the chloroplast showed unaltered transcriptional responses of water-water cycle enzymes. These findings, together with the response of marker transcripts, indicate that abscisic acid is not involved and that the plastoquinone redox state and reactive oxygen species do not play a major role in regulating the transcriptional response at t=6 h, while other marker transcripts suggest a major role for reductive power, metabolites, and lipids as signals for the response of the water-water cycle.

The plant vacuolar H(+)-ATPase takes part in acidifying compartments of the endomembrane system including the secretory pathway and the vacuoles. The structural variability of the V-ATPase complex as well as its presence in different compartments and tissues involves multiple isoforms of V-ATPase subunits. Furthermore, a versatile regulation is essential to allow for organelle- and tissue-specific fine tuning. In this study, results from V-ATPase complex disassembly with a chaotropic reagent, immunodetection and in vivo fluorescence resonance energy transfer (FRET) analyses point to a regulatory mechanism in plants, which depends on energization and involves the stability of the peripheral stalks as well. Lowering of cellular ATP by feeding 2-deoxyglucose resulted in structural alterations within the V-ATPase, as monitored by changes in FRET efficiency between subunits VHA-E and VHA-C. Potassium iodide-mediated disassembly revealed a reduced stability of V-ATPase after 2-deoxyglucose treatment of the cells, but neither the complete V(1)-sector nor VHA-C was released from the membrane in response to 2-deoxyglucose treatment, precluding a reversible dissociation mechanism like in yeast. These data suggest the existence of a regulatory mechanism of plant V-ATPase by modification of the peripheral stator structure that is linked to the cellular energization state. This mechanism is distinct from reversible dissociation as reported for the yeast V-ATPase, but might represent an evolutionary precursor of reversible dissociation.

Mitochondria from plants, yeast, and animals each contain at least one peroxiredoxin (Prx) that is involved in peroxide detoxification and redox signalling. The supramolecular dynamics of atypical type II Prx targeted to the mitochondrion was addressed in pea. Microcalorimetric (ITC) titrations identified an extremely high-affinity binding between the mitochondrial PsPrxIIF and Trx-o with a K(D) of 126+/-14 pM. Binding was driven by a favourable enthalpy change (DeltaH= -60.6 kcal mol(-1)) which was counterbalanced by unfavourable entropy changes (TDeltaS= -47.1 kcal mol(-1)). This is consistent with the occurrence of large conformational changes during binding which was abolished upon site-directed mutaganesis of the catalytic C59S and C84S. The redox-dependent interaction was confirmed by gel filtration of mitochondrial extracts and co-immunoprecipitation from extracts. The heterocomplex of PsPrxIIF and Trx-o reduced peroxide substrates more efficiently than free PsPrxIIF suggesting that Trx-o serves as an efficient and specific electron donor to PsPrxIIF in vivo. Other Trx-s tested by ITC analysis failed to interact with PsPrxIIF indicating a specific recognition of PsPrxIIF by Trx-o. PsPrxIIF exists primarily as a dimer or a hexamer depending on the redox state. In addition to the well-characterized oligomerization of classical 2-Cys Prx the results also show that atypical Prx undergo large structural reorganization with implications for protein-protein interaction and function.

The recombinant virulence protein VirE2, capable of forming a complex with single-stranded T-DNA during transfer into plant cells, was isolated, purified, and used for interactions with ssT-DNA. The in vitro interaction of VirE2 and ss-binding protein from Escherichia coli with single-stranded DNA (phage lambda) was determined by agarose gel electrophoresis by the formation of high-molecular-weight complexes after preliminary coincubation of purified protein preparations with ssDNA. We show that VirE2 binds to single-stranded DNA and protects it against nuclease S1 degradation much better than does E. coli SSB protein. We for first time observed the VirE2-ssT-DNA complex by using atomic force microscopy. The complex observed by atomic force microscopy after ssT-DNA and VirE2 protein mixing has a length of about 800 nm and a 5-8 nm width in sites with attached VirE2 protein.

In 1996, cDNA sequences referred to as plant peroxiredoxins (Prx), i.e. a 1-Cys Prx and a 2-Cys Prx, were reported from barley. Ten years of research have advanced our understanding of plant Prx as thiol-based peroxide reductases with a broad substrate specificity, ranging from hydrogen peroxide to alkyl hydroperoxides and peroxinitrite. Prx have several features in common. (i) They are abundant proteins that are routinely detected in proteomics approaches. (ii) They interact with proteins such as glutaredoxins, thioredoxins, and cyclophilins as reductants, but also non-dithiol-disulphide exchange proteins. By work with transgenic plants, their activity was shown to (iii) affect metabolic integrity, (iv) protect DNA from damage in vitro and as shown here in vivo, and (v) modulate intracellular signalling related to reactive oxygen species and reactive nitrogen species. (vi) In all organisms Prx are encoded by small gene families that are of particular complexity in higher plants. A comparison of the Prx gene families in rice and Arabidopsis thaliana supports previous suggestions on Prx function in specific subcellular and metabolic context. (vii) Prx gene expression and activity are subjected to complex regulation realized by an integration of various signalling pathways. 2-Cys Prx expression depends on redox signals, abscisic acid, and protein kinase cascades. Besides these general properties, the chloroplast Prx have acquired specific roles in the context of photosynthesis. The thioredoxin-dependent peroxidase activity can be measured in crude plant extracts and contributes significantly to the overall H(2)O(2) detoxification capacity. Thus organellar Prx proteins enable an alternative water-water cycle for detoxification of photochemically produced H(2)O(2), which acts independently from the ascorbate-dependent Asada-Halliwell-Foyer cycle. 2-Cys Prx and Prx Q associate with thylakoid membrane components. The mitochondrial PrxII F is essential for root growth under stress. Following a more general introduction, the paper summarizes present knowledge on plant organellar Prx, addressing Prx in signalling, and also suggests some lines for future research.

The genomes of the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 encode five and six open reading frames (ORFs), respectively, with similarity to peroxide-detoxifying peroxiredoxins (Prx). In addition to one highly conserved gene each for 2-Cys Prx and 1-Cys Prx, the Synechocystis sp. PCC 6803 genome contains one TypeII Prx and two PrxQ-like ORFs, while Synechococcus elongatus PCC 7942 has four PrxQ-like ORFs. The transcript regulation of all these bioinformatically identified genes was analysed under selected stress conditions, i.e. light limitation and light stress, hydrogen peroxide, methylviologen, salinity, as well as nitrogen- and iron-deficiency. The results on specific time- and stress-dependent regulation of transcript amounts suggest conserved as well as variable functions of these putative Prx-s in antioxidant defence. The results are discussed in the context of evolution and physiological function, particularly in relation to photosynthesis.

During the evolution of plants, chloroplasts have lost the exclusive genetic control over redox regulation and antioxidant gene expression. Together with many other genes, all genes encoding antioxidant enzymes and enzymes involved in the biosynthesis of low molecular weight antioxidants were transferred to the nucleus. On the other hand, photosynthesis bears a high risk for photo-oxidative damage. Concomitantly, an intricate network for mutual regulation by anthero- and retrograde signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. A major focus of recent research in chloroplast regulation addressed the mechanisms of redox sensing and signal transmission, the identification of regulatory targets, and the understanding of adaptation mechanisms. In addition to redox signals communicated through signalling cascades also used in pathogen and wounding responses, specific chloroplast signals control nuclear gene expression. Signalling pathways are triggered by the redox state of the plastoquinone pool, the thioredoxin system, and the acceptor availability at photosystem I, in addition to control by oxolipins, tetrapyrroles, carbohydrates, and abscisic acid. The signalling function is discussed in the context of regulatory circuitries that control the expression of antioxidant enzymes and redox modulators, demonstrating the principal role of chloroplasts as the source and target of redox regulation.

This work evaluates the (cor-)relations between selected biochemical responses to toxic Cd and the degree of Cd sensitivity in a set of pea genotypes. Ten genotypes were analysed that differ in their growth response to Cd when expressed as root or shoot tolerance indices (TIs). Concentrations of non-protein thiols (NPTs) and malondialdehyde (MDA), activity of chitinase, peroxidase (POX), and catalase significantly increased in all pea genotypes treated with Cd. Cd-sensitivity of genotypes was correlated with relative increases in MDA concentration as well as activities of chitinase and POX, suggesting similar Cd stress effects. Activities of ascorbate peroxidase (APX) decreased, but concentrations of glutathione (GSH) increased in the less Cd-sensitive genotypes. Differences in root and leaf contents of Cd revealed no correlation with TI, metabolic parameters, and enzyme activities in Cd-treated plants, respectively, except that shoot Cd concentration positively correlated with shoot chitinase activity. Toxic Cd levels inhibited uptake of nutrient elements such as P, K, S, Ca, Zn, Mn, and B by plants in an organ- and genotype-specific manner. Cd-sensitivity was significantly correlated with decreased root Zn concentrations. The results show both similarities, as well as distinct features, in Cd toxicity expression in genotypes of one species, suggesting that independent and multi-factorial reactions modulate Cd sensitivity on the low-tolerance level of plants. The study illustrates the biochemical basis of earlier detected genotypic variation in Cd response.

The effect of cellular p-nitrophenol (PNP) metabolism on the electro-optical (EO) characteristics of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122 was studied. When P. putida C-11 was incubated with hydroquinone, the orientational spectra of the cell suspensions changed considerably. When P. putida BA-11 and A. calcoaceticum A-122 were incubated with hydroquinone, no orientational spectrum changes were noted, possibly attesting to the operation of different PNP-metabolic pathways. In C-11, the initial metabolism of PNP may occur via the production of hydroquinone, an intermediate for PNP metabolism; in BA-11 and A. calcoaceticum A-122, via the production of 4-nitropyrocatechin, followed by a rupture of the aromatic ring. The respiratory activity of the strains toward hydroquinone was investigated concurrently. The results suggest that EO analysis is a good candidate for the study of cellular metabolism.

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions' electro-optical properties.

The electrooptical (EO) properties of a cell suspension and the specific respiratory activity of cells towards p-nitrophenol (PNP) were compared during PNP metabolism in Acinetobacter calcoaceticum strain A-122. The frequency dependence of the suspension's turbidity changes due to cellular orientation (orientational spectra) at frequencies of an orienting electric field of 10-10,000 kHz was determined. Orientational spectral changes observed during PNP incubation of the cells were followed over the range of 10-502 kHz. There were linear relationships between the magnitude of the EO effect at a 502-kHz frequency and the concentration of PNP over the range of 0.1-0.8 mM, and between the specific respiratory activity of the cells and the concentration of PNP over the range of 0.1-1.0 mM. The knowledge gained from these studies suggests a direct relationship between alterations in the cellular EO properties and PNP metabolism.

Supramembrane structures of Agrobacterium, which link cells during mating, were for the first time visualized using transmission electron microscopy. The initial cell contact was found to be mediated by long pili. Using colloidal gold-labeled, VirB1-specific antibodies, it was established that VirB1 proteins enter into the composition of short pilus-like structures, which emerge at the poles of acetosyringone (AS)-induced agrobacterial cells. Labeling of non-centrifuged agrobacterial cells on a nitrocellulose membrane using colloidal gold-conjugated antibodies to VirB1 showed that the labeled complex could bind to AS-induced cells, but failed to form red stains during incubation with cells of the Ti plasmidless A. tumefaciens strains LBA288 and UBAPF-2.

This paper reviews the results obtained in studies of the extracellular polysaccharides, lipopolysaccharide-protein complexes, polysaccharide-lipid complexes, lipopolysaccharides, and O-specific polysaccharides from bacteria of the genus Azospirillum. On the basis of present knowledge, the possible roles of the extracellular polysaccharides and polysaccharide-containing complexes of azospirilla in interaction with the roots of plants are discussed. Some pieces of evidence are considered in light of the lectin hypothesis originally proposed for the legume-Rhizobium symbiosis. In the context of these views of Azospirillumcereal associative pairs, a key process at the early stages of the interaction is the specific reaction of cereal root lectins with the extracellular polysaccharide components, containing N-acetyl-D-glucosamine as part of their structure.

Ten Azospirillum strains were serotyped using the method of cell-gold immunoblotting (dot-blot immune-overlay assay). Colloidal gold-protein A conjugate was used. Antibodies raised against the whole cells showed strain specificity and interacted mainly with carbohydrate antigens on the cell surface. Immunological identity for A. brasilense Sp 245 and Sp 107 strains was found. Cell-gold immunoblotting can be recommended for serotyping of a wide variety of bacterial strains.