Dynamics of mRNA fate during light stress and recovery: from transcription to stability and translation.

Transcript stability is an important determinant of its abundance and, consequently, translational output. Transcript destabilisation can be rapid and is well suited for modulating the cellular response. However, it is unclear the extent to which RNA stability is altered under changing environmental conditions in plants. We previously hypothesised that recovery-induced transcript destabilisation facilitated a phenomenon of rapid recovery gene downregulation (RRGD) in Arabidopsis thaliana (Arabidopsis) following light stress, based on mathematical calculations to account for ongoing transcription. Here, we test this hypothesis and investigate processes regulating transcript abundance and fate by quantifying changes in transcription, stability and translation before, during and after light stress. We adapt syringe infiltration to apply a transcriptional inhibitor to soil-grown plants in combination with stress treatments. Compared with measurements in juvenile plants and cell culture, we find reduced stability across a range of transcripts encoding proteins involved in RNA binding and processing. We also observe light-induced destabilisation of transcripts, followed by their stabilisation during recovery. We propose that this destabilisation facilitates RRGD, possibly in combination with transcriptional shut-off that was confirmed for HSP101, ROF1 and GOLS1. We also show that translation remains highly dynamic over the course of light stress and recovery, with a bias towards transcript-specific increases in ribosome association, independent of changes in total transcript abundance, after 30 min of light stress. Taken together, we provide evidence for the combinatorial regulation of transcription and stability that occurs to coordinate translation during light stress and recovery in Arabidopsis.

ConCysFind: a pipeline tool to predict conserved amino acids of protein sequences across the plant kingdom.

BACKGROUND: Post-translational modifications (PTM) of amino acid (AA) side chains in peptides control protein structure and functionality. PTMs depend on the specific AA characteristics. The reactivity of cysteine thiol-based PTMs are unique among all proteinaceous AA. This pipeline aims to ease the identification of conserved AA of polypeptides or protein families based on the phylogenetic occurrence in the plant kingdom. The tool is customizable to include any species. The degree of AA conservation is taken as indicator for structural and functional significance, especially for PTM-based regulation. Further, this pipeline tool gives insight into the evolution of these potentially regulatory important peptides. RESULTS: The web-based or stand-alone pipeline tool Conserved Cysteine Finder (ConCysFind) was developed to identify conserved AA such as cysteine, tryptophan, serine, threonine, tyrosin and methionine. ConCysFind evaluates multiple alignments considering the proteome of 21 plant species. This exemplar study focused on Cys as evolutionarily conserved target for multiple redox PTM. Phylogenetic trees and tables with the compressed results of the scoring algorithm are generated for each Cys in the query polypeptide. Analysis of 33 translation elongation and release factors alongside of known redox proteins from Arabidopsis thaliana for conserved Cys residues confirmed the suitability of the tool for identifying conserved and functional PTM sites. Exemplarily, the redox sensitivity of cysteines in the eukaryotic release factor 1-1 (eRF1-1) was experimentally validated. CONCLUSION: ConCysFind is a valuable tool for prediction of new potential protein PTM targets in a broad spectrum of species, based on conserved AA throughout the plant kingdom. The identified targets were successfully verified through protein biochemical assays. The pipeline is universally applicable to other phylogenetic branches by customization of the database.

Phytoglobins in the nuclei, cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid.

Symbiotic hemoglobins provide O2 to N2 -fixing bacteria within legume nodules, but the functions of non-symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C-terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S-nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S-nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA-Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.

Redox and Reactive Oxygen Species Network in Acclimation for Salinity Tolerance in Sugar Beet.

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.

Redox Regulation of Cytosolic Translation in Plants.

Control of protein homeostasis is crucial for environmental acclimation of plants. In this context, translational control is receiving increasing attention, particularly since post-translational modifications of the translational apparatus allow very fast and highly effective control of protein synthesis. Reduction and oxidation (redox) reactions decisively control translation by modifying initiation, elongation, and termination of translation. This opinion article compiles information on the redox sensitivity of cytosolic translation factors and the significance of redox regulation as a key modulator of translation for efficient acclimation to changing environmental conditions.

Function of glutathione peroxidases in legume root nodules.

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.

Assessing redox state and reactive oxygen species in circadian rhythmicity.

Redox homeostasis is an important parameter of cell function and cell signaling. Spatial and temporal alterations of redox state control metabolism, developmental processes, as well as acute responses to environmental stresses and stress acclimation. Redox homeostasis is also linked to the circadian clock. This chapter introduces methods to assess important redox parameters such as the low molecular weight redox metabolites glutathione and ascorbate, their amount and redox state, and H2O2 as reactive oxygen species. In vivo redox cell imaging is described by use of the reduction-oxidation sensitive green fluorescent protein (roGFP). Finally, on the level of posttranslational redox modifications of proteins, methods are shown to assess hyperoxidation of 2-cysteine peroxiredoxin and glutathionylation of peroxiredoxin IIE. The redox state of 2-cysteine peroxiredoxin has been identified as a transcription-independent marker of circadian rhythmicity.

Kinetics of retrograde signalling initiation in the high light response of Arabidopsis thaliana.

High light acclimation depends on retrograde control of nuclear gene expression. Retrograde regulation uses multiple signalling pathways and thus exploits signal patterns. To maximally challenge the acclimation system, Arabidopsis thaliana plants were either adapted to 8 (low light (L-light)) or 80 µmol quanta m(-2) s(-1) (normal light (N-light)) and subsequently exposed to a 100- and 10-fold light intensity increase, respectively, to high light (H-light, 800 µmol quanta m(-2) s(-1)), for up to 6 h. Both L → H- and N → H-light plants efficiently regulated CO2 assimilation to a constant level without apparent damage and inhibition. This experimental set-up was scrutinized for time-dependent regulation and efficiency of adjustment. Transcriptome profiles revealed that N-light and L-light plants differentially accumulated 2119 transcripts. After 6 h in H-light, only 205 remained differently regulated between the L → H- and N → H-light plants, indicating efficient regulation allowing the plants to reach a similar transcriptome state. Time-dependent analysis of transcripts as markers for signalling pathways, and of metabolites and hormones as possibly involved transmitters, suggests that oxylipins such as oxophytodienoic acid and jasmonic acid, metabolites and redox cues predominantly control the acclimation response, whereas abscisic acid, salicylic acid and auxins play an insignificant or minor role.

Fast retrograde signaling in response to high light involves metabolite export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF transcription factors in Arabidopsis.

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.