The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo

Muthuramalingam M, Matros A, Scheibe R, Mock H-P, Dietz K-J. The hydrogen peroxide-sensitive proteome of the chloroplast in vitro and in vivo. Frontiers in Plant Science. 2013;4:54-1-54-14.Hydrogen peroxide (H2O2) evolves during cellular metabolism and accumulates under various stresses causing serious redox imbalances. Many proteomics studies aiming to identify proteins sensitive to H2O2 used concentrations that were above the physiological range. Here the chloroplast proteins were subjected to partial oxidation by exogenous addition of H2O2 equivalent to 10% of available protein thiols which allowed for the identification of the primary targets of oxidation. The chosen redox proteomic approach employed differential labeling of non-oxidized and oxidized thiols using sequential alkylation with N-ethylmaleimide and biotin maleimide. The in vitro identified proteins are involved in carbohydrate metabolism, photosynthesis, redox homeostasis, and nitrogen assimilation. By using methyl viologen that induces oxidative stress in vivo, mostly the same primary targets of oxidation were identified and several oxidation sites were annotated. Ribulose-1,5-bisphosphate (RubisCO) was a primary oxidation target. Due to its high abundance, RubisCO is suggested to act as a chloroplast redox buffer to maintain a suitable redox state, even in the presence of increased reactive oxygen species release. 2-cysteine peroxiredoxins (2-Cys Prx) undergo redox-dependent modifications and play important roles in antioxidant defense and signaling. The identification of 2-Cys Prx was expected based on its high affinity to H2O2 and is considered as a proof of concept for the approach. Targets of Trx, such as phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, transketolase, and sedoheptulose-1,7-bisphosphatase have at least one regulatory disulfide bridge which supports the conclusion that the identified proteins undergo reversible thiol oxidation. In conclusion, the presented approach enabled the identification of early targets of H2O2 oxidation within the cellular proteome under physiological experimental conditions

The low molecular weight fraction of compounds released from immature wheat pistils supports barley pollen embryogenesis

Pollen embryogenesis provides a useful means of generating haploid plants for plant breeding and basic research. Although it is well established that the efficacy of the process can be enhanced by the provision of immature pistils as a nurse tissue, the origin and compound class of the signal molecule(s) involved is still elusive. Here, a micro-culture system was established to enable the culturing of populations of barley pollen at a density too low to allow unaided embryogenesis to occur, and this was then exploited to assess the effect of using various parts of the pistil as nurse tissue. A five fold increase in the number of embryogenic calli formed was obtained by simply cutting the pistils in half. The effectiveness of the pistil-conditioned medium was transitory, since it needed replacement at least every four days to measurably ensure embryogenic development. The differential effect of various size classes of compounds present in the pistil-conditioned medium showed that the relevant molecule(s) was of molecular weight below 3 kDa. This work narrows down possible feeder molecules to lower molecular weight compounds and showed that the cellular origin of the active compound(s) is not specific to any tested part of the pistil. Furthermore the increased recovery of calli during treatment with cut pistils may provide a useful tool for plant breeders and researchers using haploid technology in barley and other plant species

Comparative evaluation of extraction methods for apoplastic proteins from maize leaves

Proteins in the plant apoplast are essential for many physiological processes. We have analysed and compared six different infiltration solutions for proteins contained in the apoplast to recognize the most suitable method for leaves and to establish proteome maps for each extraction. The efficiency of protocols was evaluated by comparing the protein patterns resolved by 1-DE and 2-DE, and revealed distinct characteristics for each infiltration solution. Nano-LC-ESI-Q-TOF MS analysis of all fractions was applied to cover all proteins differentially extracted by infiltration solutions and led to the identification of 328 proteins in total in apoplast preparations. The predicted subcellular protein localisation distinguished the examined infiltration solutions in those with high or low amounts of intracellular protein contaminations, and with high or low quantities of secreted proteins. All tested infiltration solution extracted different subsets of proteins, and those implications on apoplast-specific studies are discussed

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development

Lincomycin (LIN)‐mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis‐related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de‐repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole‐genome single‐nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell‐wall mutants establishes a link between secondary cell‐wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing

Function and Regulation of Chloroplast Peroxiredoxin IIE

Peroxiredoxins (PRX) are thiol peroxidases that are highly conserved throughout all biological kingdoms. Increasing evidence suggests that their high reactivity toward peroxides has a function not only in antioxidant defense but in particular in redox regulation of the cell. Peroxiredoxin IIE (PRX-IIE) is one of three PRX types found in plastids and has previously been linked to pathogen defense and protection from protein nitration. However, its posttranslational regulation and its function in the chloroplast protein network remained to be explored. Using recombinant protein, it was shown that the peroxidatic Cys121 is subjected to multiple posttranslational modifications, namely disulfide formation, S-nitrosation, S-glutathionylation, and hyperoxidation. Slightly oxidized glutathione fostered S-glutathionylation and inhibited activity in vitro. Immobilized recombinant PRX-IIE allowed trapping and subsequent identification of interaction partners by mass spectrometry. Interaction with the 14-3-3 υ protein was confirmed in vitro and was shown to be stimulated under oxidizing conditions. Interactions did not depend on phosphorylation as revealed by testing phospho-mimicry variants of PRX-IIE. Based on these data it is proposed that 14-3-3υ guides PRX‑IIE to certain target proteins, possibly for redox regulation. These findings together with the other identified potential interaction partners of type II PRXs localized to plastids, mitochondria, and cytosol provide a new perspective on the redox regulatory network of the cell

Combined nitrogen and drought stress leads to overlapping and unique proteomic responses in potato

Main conclusion: Nitrogen deficient and drought-tolerant or sensitive potatoes differ in proteomic responses under combined (NWD) and individual stresses. The sensitive genotype ‘Kiebitz’ exhibits a higher abundance of proteases under NWD. Abstract: Abiotic stresses such as N deficiency and drought affect the yield of Solanum tuberosum L. tremendously. Therefore, it is of importance to improve potato genotypes in terms of stress tolerance. In this study, we identified differentially abundant proteins (DAPs) in four starch potato genotypes under N deficiency (ND), drought stress (WD), or combined stress (NWD) in two rain-out shelter experiments. The gel-free LC–MS analysis generated a set of 1177 identified and quantified proteins. The incidence of common DAPs in tolerant and sensitive genotypes under NWD indicates general responses to this stress combination. Most of these proteins were part of the amino acid metabolism (13.9%). Three isoforms of S-adenosyl methionine synthase (SAMS) were found to be lower abundant in all genotypes. As SAMS were found upon application of single stresses as well, these proteins appear to be part of the general stress response in potato. Interestingly, the sensitive genotype ‘Kiebitz’ showed a higher abundance of three proteases (subtilase, carboxypeptidase, subtilase family protein) and a lower abundance of a protease inhibitor (stigma expressed protein) under NWD stress compared to control plants. The comparably tolerant genotype ‘Tomba’, however, displayed lower abundances of proteases. This indicates a better coping strategy for the tolerant genotype and a quicker reaction to WD when previously stressed with ND

A transposon-based activation-tagging population in Arabidopsis thaliana (TAMARA) and its application in the identification of dominant developmental and metabolic mutations

AbstractA population of 9471 stable activation-tagged lines was generated by transposable element mediated activation tagging mutagenesis in Arabidopsis (TAMARA) using the maize En/Spm transposon system. Based on DNA gel blot and flanking sequence analysis, this population contains approximately 6000 independent transposon insertions. A greenhouse-based screen identified six dominant or semi-dominant activation tagged mutants with obvious developmental alterations, among these a new pistillata mutant allele. In addition, a subset of 1500 lines was screened by a HPLC based high-throughput method for dominant activation tagged mutants with enhanced contents of phenolic compounds. One dominant activation tagged mutant (hpc1-1D) was isolated showing accumulation of a particular compound due to the upregulation of an R2R3-MYB transcription factor

Protein Composition and Baking Quality of Wheat Flour as Affected by Split Nitrogen Application

Baking quality of wheat flour is determined by grain protein concentration (GPC) and its composition and is highly influenced by environmental factors such as nitrogen (N) fertilization management. This study investigated the effect of split N application on grain protein composition and baking quality of two winter wheat cultivars, Tobak and JB Asano, belonging to different baking quality classes. Bread loaf volumes in both cultivars were enhanced by split N application. In contrast, GPC was only significantly increased in JB Asano. Comparative 2-DE revealed that the relative volumes of 21 and 28 unique protein spots were significantly changed by split N application in Tobak and JB Asano, respectively. Specifically, the alterations in relative abundance of certain proteins, i.e., globulins, LMW-GS, α-, and γ-gliadins as well as α-amylase/trypsin inhibitors were more sensitive to split N application. Furthermore, certain proteins identified as globulins and alpha-amylase inhibitors were changed in both wheat cultivars under split N application. These results implied that the functions of these unique proteins might have played important roles in affecting baking quality of wheat flour, especially for cultivars (i.e., Tobak in the present study) the baking quality of which is less dependent on GPC