Arabidopsis Thaliana Cell Suspension Research Articles

A combined lipidomic and proteomic profiling of Arabidopsis thaliana plasma membrane.

The plant plasma membrane (PM) plays a key role in perception of environmental signals, and set-up of adaptive responses. An exhaustive and quantitative description of the whole set of lipids and proteins constituting the PM is necessary to understand how these components allow to fulfill such essential physiological functions. Here we provide by state-of-the-art approaches the first combined reference of the plant PM lipidome and proteome from Arabidopsis thaliana suspension cell culture. We identified and quantified a reproducible core set of 2165 proteins, which is by far the largest set of available data concerning this plant PM proteome. Using the same samples, combined lipidomic approaches, allowing the identification and quantification of an unprecedented repertoire of 414 molecular species of lipids showed that sterols, phospholipids, and sphingolipids are present in similar proportions in the plant PM. Within each lipid class, the precise amount of each lipid family and the relative proportion of each molecular species were further determined, allowing to establish the complete lipidome of Arabidopsis PM, and highlighting specific characteristics of the different molecular species of lipids. Results obtained point to a finely tuned adjustment of the molecular characteristics of lipids and proteins. More than a hundred proteins related to lipid metabolism, transport, or signaling have been identified and put in perspective of the lipids with which they are associated. This set of data represents an innovative resource to guide further research relative to the organization and functions of the plant PM.

In Vivo Phosphorylation of the Cytosolic Glucose-6-Phosphate Dehydrogenase Isozyme G6PD6 in Phosphate-Resupplied Arabidopsis thaliana Suspension Cells and Seedlings.

Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first committed step of the oxidative pentose phosphate pathway (OPPP). Our recent phosphoproteomics study revealed that the cytosolic G6PD6 isozyme became hyperphosphorylated at Ser12, Thr13 and Ser18, 48 h following phosphate (Pi) resupply to Pi-starved (-Pi) Arabidopsis thaliana cell cultures. The aim of the present study was to assess whether G6PD6 phosphorylation also occurs in shoots or roots following Pi resupply to -Pi Arabidopsis seedlings, and to investigate its relationship with G6PD activity. Interrogation of phosphoproteomic databases indicated that N-terminal, multi-site phosphorylation of G6PD6 and its orthologs is quite prevalent. However, the functions of these phosphorylation events remain unknown. Immunoblotting with an anti-(pSer18 phosphosite-specific G6PD6) antibody confirmed that G6PD6 from Pi-resupplied, but not -Pi, Arabidopsis cell cultures or seedlings (i.e., roots) was phosphorylated at Ser18; this correlated with a significant increase in extractable G6PD activity, and biomass accumulation. Peptide kinase assays of Pi-resupplied cell culture extracts indicated that G6PD6 phosphorylation at Ser18 is catalyzed by a Ca2+-dependent protein kinase (CDPK), which correlates with the 'CDPK-like' targeting motif that flanks Ser18. Our results support the hypothesis that N-terminal phosphorylation activates G6PD6 to enhance OPPP flux and thus the production of reducing power (i.e., NADPH) and C-skeletons needed to establish the rapid resumption of growth that ensures Pi-resupply to -Pi Arabidopsis.

Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant-programmed cell death.

Programmed cell death (PCD) facilitates selective, genetically controlled elimination of redundant, damaged, or infected cells. In plants, PCD is often an essential component of normal development and can mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of PCD is hindered by difficulties in sampling small groups of dying cells that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing and Arabidopsis thaliana suspension cells, a model system that allows precise monitoring of PCD rates. The use of three PCD-inducing treatments (salicylic acid, heat, and critical dilution), in combination with three cell death modulators (3-methyladenine, lanthanum chloride, and conditioned medium), enabled isolation of candidate core- and stimuli-specific PCD genes, inference of underlying regulatory networks and identification of putative transcriptional regulators of PCD in plants. This analysis underscored a disturbance of the cell cycle and mitochondrial retrograde signaling, and repression of pro-survival stress responses, as key elements of the PCD-associated transcriptional signature. Further, phenotyping of Arabidopsis T-DNA insertion mutants in selected candidate genes validated the potential of generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance.

The boundary of life and death: changes in mitochondrial and cytosolic proteomes associated with programmed cell death of Arabidopsis thaliana suspension culture cells.

Despite the critical role of programmed cell death (PCD) in plant development and defense responses, its regulation is not fully understood. It has been proposed that mitochondria may be important in the control of the early stages of plant PCD, but the details of this regulation are currently unknown. We used Arabidopsis thaliana cell suspension culture, a model system that enables induction and precise monitoring of PCD rates, as well as chemical manipulation of this process to generate a quantitative profile of the alterations in mitochondrial and cytosolic proteomes associated with early stages of plant PCD induced by heat stress. The cells were subjected to PCD-inducing heat levels (10 min, 54°C), with/without the calcium channel inhibitor and PCD blocker LaCl3. The stress treatment was followed by separation of cytosolic and mitochondrial fractions and mass spectrometry-based proteome analysis. Heat stress induced rapid and extensive changes in protein abundance in both fractions, with release of mitochondrial proteins into the cytosol upon PCD induction. In our system, LaCl3 appeared to act downstream of cell death initiation signal, as it did not affect the release of mitochondrial proteins, but instead partially inhibited changes occurring in the cytosolic fraction, including upregulation of proteins with hydrolytic activity. We characterized changes in protein abundance and localization associated with the early stages of heat stress-induced PCD. Collectively, the generated data provide new insights into the regulation of cell death and survival decisions in plant cells.

CRISPR/Cas9-Mediated Targeted DNA Integration: Rearrangements at the Junction of Plant and Plasmid DNA.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.

Protoplast Preparation and Fluorescence-Activated Cell Sorting for the Evaluation of Targeted Mutagenesis in Plant Cells.

Fluorescence-activated cell sorting (FACS) allows for the enrichment of specific plant cell populations after protoplasting. In this book chapter, we describe the transformation and protoplasting of an Arabidopsis thaliana cell suspension culture (PSB-D, derived from MM2d) that can be used for the evaluation of CRISPR vectors in a subpopulation of cells. We also describe the protoplasting of Arabidopsis thaliana cells from the roots and stomatal lineage for the evaluation of tissue-specific gene editing. These protocols allow us to rapidly and accurately quantify various CRISPR systems in plant cells.

Chemical Perturbation of Chloroplast Ca2+ Dynamics in Arabidopsis thaliana Suspension Cell Cultures and Seedlings.

Ca2+ signaling is part of universal signal transduction pathways to respond to external and internal stimuli or stress and in plants plays a central role in chloroplasts, such as in the regulation of photosynthetic enzymes or the transition from light to dark. Only recently, the underlying molecular machinery, e.g., transporters and channels that enable chloroplast Ca2+ fluxes, has started to be elucidated. However, chemical tools to specifically perturb these chloroplast Ca2+ fluxes are largely lacking. Here, we describe an efficient aequorin-based system in Arabidopsis thaliana suspension cell cultures to screen for chemicals that alter light-to-dark-induced chloroplast stroma Ca2+ signals. Subsequently, the effect of the hits on chloroplast Ca2+ signals is validated in Arabidopsis seedlings. The research lays a foundation for the identification of novel proteins involved in Ca2+ transport in chloroplast stroma under light-to-dark transition and for investigating the interaction of chloroplast Ca2+ signaling with photosynthesis in general.

Arabidopsis PAP17 is a dual-localized purple acid phosphatase up-regulated during phosphate deprivation, senescence, and oxidative stress

A 35 kDa monomeric purple acid phosphatase (APase) was purified from cell wall extracts of Pi starved (-Pi) Arabidopsis thaliana suspension cells and identified as AtPAP17 (At3g17790) by mass spectrometry and N-terminal microsequencing. AtPAP17 was de novo synthesized and dual-localized to the secretome and/or intracellular fraction of -Pi or salt-stressed plants, or senescing leaves. Transiently expressed AtPAP17-green fluorescent protein localized to lytic vacuoles of the Arabidopsis suspension cells. No significant biochemical or phenotypical changes associated with AtPAP17 loss of function were observed in an atpap17 mutant during Pi deprivation, leaf senescence, or salinity stress. Nevertheless, AtPAP17 is hypothesized to contribute to Pi metabolism owing to its marked up-regulation during Pi starvation and leaf senescence, broad APase substrate selectivity and pH activity profile, and rapid repression and turnover following Pi resupply to -Pi plants. While AtPAP17 also catalyzed the peroxidation of luminol, which was optimal at pH 9.2, it exhibited a low Vmax and affinity for hydrogen peroxide relative to horseradish peroxidase. These results, coupled with absence of a phenotype in the salt-stressed or -Pi atpap17 mutant, do not support proposals that the peroxidase activity of AtPAP17 contributes to the detoxification of reactive oxygen species during stresses that trigger AtPAP17 up-regulation.

Stable Transformation of Arabidopsis thaliana Cell Suspension Cultures: A Case Study for The Overexpression of the COI1 Receptor.

Cell suspension cultures have been studied for decades to produce natural molecules. However, the difficulty in generating stably transformed cell lines has limited their use to produce high value chemicals reproducibly and in elevated quantities. In this protocol, a method to stably transform and maintain Arabidopsis cell suspension cultures is devised and presented in detail. Arabidopsis cell cultures were directly transformed with A. tumefaciens for the overexpression of the CORONATINE INSENSITIVE 1 (COI1) jasmonate receptor. Cell cultures were established after transformation and continuously maintained and tested for the overexpression of COI1. The protocol was also previously used to silence Arabidopsis peroxidases and allows for long term maintenance of transformed cells. Details on culture maintenance, both in liquid and solid media are provided, alongside with evidence of protein expression to confirm transformation. The system described provides a powerful tool for synthetic biology to study signaling independent of developmental control and to obtain metabolites of interest for the biotechnological and medical sectors.

The cyanobacterium, Nostoc punctiforme can protect against programmed cell death and induce defence genes in Arabidopsis thaliana

ABSTRACT Cyanobacteria can form nitrogen-fixing symbioses with a broad range of plant species. Unlike other plant-bacteria symbioses, little is understood about the immunological responses induced by plant cyanobionts (symbiotic cyanobacteria). Here, we demonstrated that the model plant-symbiotic cyanobacteria, Nostoc punctiforme is capable of protecting against programmed cell death (PCD) when induced in Arabidopsis thaliana suspension cell cultures. We also profiled the early transcriptomic changes that were induced in response to conditioned medium (CM) from N. punctiforme cell cultures. Interestingly, the reduction in PCD was preceded by the induction of genes associated with defence and immunity, the most striking of which were a number of WRKY-family transcription factors. Down-regulated genes included those involved in the regulation of cell growth and differentiation. This work is the first to show that a cyanobiont can affect plant PCD and provides a useful transcriptome resource for studying early plant cell responses to symbiotic cyanobacteria.

Phosphate and phosphite have a differential impact on the proteome and phosphoproteome of Arabidopsis suspension cell cultures.

Phosphorus absorbed in the form of phosphate (H2 PO4- ) is an essential but limiting macronutrient for plant growth and agricultural productivity. A comprehensive understanding of how plants respond to phosphate starvation is essential for the development of more phosphate-efficient crops. Here we employed label-free proteomics and phosphoproteomics to quantify protein-level responses to 48h of phosphate versus phosphite (H2 PO3- ) resupply to phosphate-deprived Arabidopsis thaliana suspension cells. Phosphite is similarly sensed, taken up and transported by plant cells as phosphate, but cannot be metabolized or used as a nutrient. Phosphite is thus a useful tool for differentiating between non-specific processes related to phosphate sensing and transport and specific responses to phosphorus nutrition. We found that responses to phosphate versus phosphite resupply occurred mainly at the level of protein phosphorylation, complemented by limited changes in protein abundance, primarily in protein translation, phosphate transport and scavenging, and central metabolism proteins. Altered phosphorylation of proteins involved in core processes such as translation, RNA splicing and kinase signaling was especially important. We also found differential phosphorylation in response to phosphate and phosphite in 69 proteins, including splicing factors, translation factors, the PHT1;4 phosphate transporter and the HAT1 histone acetyltransferase - potential phospho-switches signaling changes in phosphorus nutrition. Our study illuminates several new aspects of the phosphate starvation response and identifies important targets for further investigation and potential crop improvement.

Auxin protects Arabidopsis thaliana cell suspension cultures from programmed cell death induced by the cellulose biosynthesis inhibitors thaxtomin A and isoxaben

BackgroundThaxtomin A (TA) is a natural cellulose biosynthesis inhibitor (CBI) synthesized by the potato common scab-causing pathogen Streptomyces scabies. Inhibition of cellulose synthesis by TA compromises cell wall organization and integrity, leading to the induction of an atypical program of cell death (PCD). These processes may facilitate S. scabies entry into plant tissues. To study the mechanisms that regulate the induction of cell death in response to inhibition of cellulose synthesis, we used Arabidopsis thaliana cell suspension cultures treated with two structurally different CBIs, TA and the herbicide isoxaben (IXB).ResultsThe induction of cell death by TA and IXB was abrogated following pretreatment with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the natural auxin indole-3-acetic acid (IAA). The addition of auxin efflux inhibitors also inhibited the CBI-mediated induction of PCD. This effect may be due to intracellular accumulation of auxin. Auxin has a wide range of effects in plant cells, including a role in the control of cell wall composition and rigidity to facilitate cell elongation. Using Atomic Force Microscopy (AFM)-based force spectroscopy, we found that inhibition of cellulose synthesis by TA and IXB in suspension-cultured cells decreased cell wall stiffness to a level slightly different than that caused by auxin. However, the cell wall stiffness in cells pretreated with auxin prior to CBI treatment was equivalent to that of cells treated with auxin only.ConclusionsAddition of auxin to Arabidopsis cell suspension cultures prevented the TA- and IXB-mediated induction of cell death. Cell survival was also stimulated by inhibition of polar auxin transport during CBI-treatment. Inhibition of cellulose synthesis perturbed cell wall mechanical properties of Arabidopsis cells. Auxin treatment alone or with CBI also decreased cell wall stiffness, showing that the mechanical properties of the cell wall perturbed by CBIs were not restored by auxin. However, since auxin’s effects on the cell wall stiffness apparently overrode those induced by CBIs, we suggest that auxin may limit the impact of CBIs by restoring its own transport and/or by stabilizing the plasma membrane - cell wall - cytoskeleton continuum.

Structure and Activity of a Cytosolic Ribosome-Inactivating Protein from Rice.

Ribosome-inactivating proteins (RIPs) are cytotoxic enzymes that inhibit protein translation by depurinating ribosomal RNA. Although most plant RIPs are synthesized with leader sequences that sequester them away from the host ribosomes, several RIPs from cereals lack these signal peptides and therefore probably reside in the cytosol near the plant ribosomes. More than 30 RIP genes have been identified in the rice (Oryza sativa spp. japonica) genome, many of them lacking a signal peptide. This paper focuses on a presumed cytosolic type-1 RIP from rice, referred to as OsRIP1. Using 3D modeling it is shown that OsRIP1 structurally resembles other cereal RIPs and has an active site that meets the requirements for activity. Furthermore, localization studies indicate that OsRIP1-eGFP fusion proteins reside in the nucleocytoplasmic space when expressed in epidermal cells of Nicotiana benthamiana or Arabidopsis thaliana suspension cells. Finally, OsRIP1 was recombinantly produced in Escherichia coli and was demonstrated to possess catalytic activity. Interestingly, this recombinant RIP inactivates wheat ribosomes far less efficiently than rabbit ribosomes in an in vitro system. These findings raise some interesting questions concerning the mode of action and physiological role of OsRIP1. This is the first time a RIP from rice is investigated at protein level and is shown to possess biological activity.

Heat stress reveals high molecular mass proteasomes in Arabidopsis thaliana suspension cells cultures

Because of their sessile nature, plants have evolved complex and robust mechanisms to respond to adverse environments. Stress conditions trigger an increase in protein turnover and degradation. Proteasomes are essential to the cell for removing, in a highly regulated manner, partially denatured or oxidized proteins thus minimizing their cytotoxicity. We observed that suspension cells of Arabidopsis thaliana treated with high temperature (37 °C) directed the assembly of high molecular mass proteasomes. The removal of a 75% of the original ubiquitin conjugates and the maintenance of protein carbonyls at basal levels correlated with a specific proteasome profiles. The profiles obtained by the separation of different proteasomes populations by Blue-Native Polyacrylamide Gel Electrophoresis and western blot analysis suggest that synthesis, assembly, and heavy ubiquitination of 20S (CP) subunits are promoted by heat stress.

Drought stress induces a biphasic NO accumulation in Arabidopsis thaliana

ABSTRACTWe have recently reported the proteomic signature of the early (≤30 min) drought stress responses in Arabidopsis thaliana suspension cells challenged with PEG. We found an over-representation in the gene ontology categories “Ribosome” and “Oxidative stress along with an increased abundance of late embryogenesis abundant (LEA) and early response to dehydration (ERD) proteins. Since nitric oxide (NO) plays a pivotal role in plant responses to drought stress and induces LEA and DREB proteins, here we monitored the levels of NO in Arabidopsis cell suspensions and leaf disks challenged with PEG, and performed comparative analyses of the proteomics and transcriptomics data in public domain to search for a common set of early drought and NO responsive proteins.We show that under drought-stress, NO shows a biphasic time course, much like in response to ozone stress and that among the early drought and NO responsive proteins, the categories “DNA binding”, “Nucleotide binding” and “Transcription regulator activity” are enriched. Taken together, present study suggests that in Arabidopsis the changing NO levels may play a critical role in early drought responsive processes and notably in the transcriptional and translational reprograming observed under drought stress.

Early Responses to Severe Drought Stress in the Arabidopsis thaliana Cell Suspension Culture Proteome.

Abiotic stresses are considered the most deleterious factor affecting growth and development of plants worldwide. Such stresses are largely unavoidable and trigger adaptive responses affecting different cellular processes and target different compartments. Shotgun proteomic and mass spectrometry-based approaches offer an opportunity to elucidate the response of the proteome to abiotic stresses. In this study, the severe drought or water-deficit response in Arabidopsis thaliana was mimicked by treating cell suspension callus with 40% polyethylene glycol for 10 and 30 min. Resulting data demonstrated that 310 proteins were differentially expressed in response to this treatment with a strict ±2.0-fold change. Over-representation was observed in the gene ontology categories of ‘ribosome’ and its related functions as well as ‘oxidative phosphorylation’, indicating both structural and functional drought responses at the cellular level. Proteins in the category ‘endocytosis’ also show significant enrichment and this is consistent with increased active transport and recycling of membrane proteins in response to abiotic stress. This is supported by the particularly pronounced enrichment in proteins of the endosomal sorting complexes that are required for membrane remodelling. Taken together, the findings point to rapid and complex physiological and structural changes essential for survival in response to sudden severe drought stress.

Design, synthesis and perception of fluorescently labeled isoprenoid cytokinins

Isoprenoid cytokinins play a number of crucial roles in the regulation of plant growth and development. To study cytokinin receptor properties in plants, we designed and prepared fluorescent derivatives of 6-[(3-methylbut-2-en-1-yl)amino]purine (N6-isopentenyladenine, iP) with several fluorescent labels attached to the C2 or N9 atom of the purine moiety via a 2- or 6-carbon linker. The fluorescent labels included dansyl (DS), fluorescein (FC), 7-nitrobenzofurazan (NBD), rhodamine B (RhoB), coumarin (Cou), 7-(diethylamino)coumarin (DEAC) and cyanine 5 dye (Cy5). All prepared compounds were screened for affinity for the Arabidopsis thaliana cytokinin receptor (CRE1/AHK4). Although the attachment of the fluorescent labels to iP via the linkers mostly disrupted binding to the receptor, several fluorescent derivatives interacted well. For this reason, three derivatives, two rhodamine B and one 4-chloro-7-nitrobenzofurazan labeled iP were tested for their interaction with CRE1/AHK4 and Zea mays cytokinin receptors in detail. We further showed that the three derivatives were able to activate transcription of cytokinin response regulator ARR5 in Arabidopsis seedlings. The activity of fluorescently labeled cytokinins was compared with corresponding 6-dimethylaminopurine fluorescently labeled negative controls. Selected rhodamine B C2-labeled compounds 17, 18 and 4-chloro-7-nitrobenzofurazan N9-labeled compound 28 and their respective negative controls (19, 20 and 29, respectively) were used for in planta staining experiments in Arabidopsis thaliana cell suspension culture using live cell confocal microscopy.

Identification of Tyrosine and Nitrotyrosine with a Mixed-Mode Solid-Phase Extraction Cleanup Followed by Liquid Chromatography-Electrospray Time-of-Flight Mass Spectrometry in Plants.

In higher plants, there is a growing interest in the study of protein tyrosine nitration (NO2Tyr) as well as the identification of in vivo nitrated proteins. Different methods have been developed for identifying nitrotyrosine in biological samples. However, these analyses are difficult because tyrosine nitration is a very low-abundance posttranslational protein modification (PTM) and the lack of efficient enrichment methods for detection. The identification and quantification of NO2Tyr in proteins has represented a challenge for researchers.In this chapter a new method for determining NO2Tyr and tyrosine (Tyr) in Arabidopsis thaliana cell-suspension culture extracts is proposed. The quantification was performed using a simple, sensitive, and specific sample preparation assay based on mixed-mode solid-phase extraction (SPE) which was developed for the quantification of trace NO2Tyr in Arabidopsis extracts by liquid chromatography-electrospray time-of-flight mass spectrometry (LC-TOFMS).

Expression analysis of transporter genes for screening candidate monolignol transporters using Arabidopsis thaliana cell suspensions during tracheary element differentiation.

The mechanism of monolignol transportation from the cytosol to the apoplast is still unclear despite being an essential step of lignification. Recently, ATP-binding cassette (ABC) transporters were suggested to be involved in monolignol transport. However, there are no reliable clues to the transporters of the major lignin monomers coniferyl and synapyl alcohol. In this study, the lignification progress of Arabidopsis cultured cells during tracheary element differentiation was monitored. The expression of selected transporter genes, as well as lignification and cell-wall formation related genes as references, in differentiating cultured cell samples harvested at 2-day intervals was analyzed by real-time PCR and the data were statistically processed. The cell wall formation transcription factor MYB46, programmed-cell death related gene XCP1 and lignin polymerization peroxidase AtPrx25 were classified into the same cluster. Furthermore, the cluster closest to the abovementioned cluster contained the lignin synthesis transcription factor MYB58 and the Arabidopsis ABC transporters ABCG11, ABCG22, ABCG36 and ABCG29. This result suggested that these four ABC transporters may be involved in lignification. In the expression analysis, unexpectedly, the lignification-related genes CAD5 and C4H were not included in the same cluster as MYB58 and AtPrx25. The expression data also suggested that the lignification of tracheary elements in the culture, where lignification ratio finally reached to around 40%, continued after cell death because lignification actively progressed after programmed cell death-related gene started to be expressed.

Bicarbonate Induced Redox Proteome Changes in Arabidopsis Suspension Cells.

Climate change as a result of increasing atmospheric CO2 affects plant growth and productivity. CO2 is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO2 response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in Arabidopsis thaliana suspension cells in response to bicarbonate (mimic of elevated CO2) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO2 responses.