The thioredoxin h-type TdTrxh2 protein of durum wheat confers abiotic stress tolerance of the transformant Arabidopsis plants through its protective role and the regulation of redox homoeostasis.

The thioredoxins (Trxs) are ubiquitous and they play a crucial role in various biological processes like growth and stress response. Although the functions of Trxs proteins are described in several previous reports, the function of the isoform Trxh2 of durum wheat (Triticum durum L.), designated as TdTrxh2, in abiotic stress response still unknown. Thus, we aimed in this study the functional characterization of TdTrxh2 through its expression in yeast cells and Arabidopsis plants. Sequence analysis revealed that TdTrxh2 protein shared the conserved redox site with the other Trxh from other plant species. Under various abiotic stresses, TdTrxh2 was up-regulated in leaves and roots of durum wheat. Interestingly, we demonstrated that TdTrxh2 exhibit protective effect on LDH activity against various treatments. Besides, the expression of TdTrxh2 in yeast cells conferred their tolerance to multiple stresses. Moreover, transgenic Arabidopsis expressing TdTrxh2 showed tolerance phenotype to several abiotic stresses. This tolerance was illustrated by high rate of proline accumulation, root proliferation, low accumulation of reactive oxygen species like H2O2 and O2·-, and high antioxidant CAT and POD enzymes activities. All these findings suggested that TdTrxh2 promotes abiotic stress tolerance through the redox homoeostasis regulation and its protective role.

Barley stripe mosaic virus γb protein targets thioredoxin h-type 1 to dampen salicylic acid-mediated defenses.

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction, and how plant viruses manipulate the SA-dependent defense responses requires further characterization. Here, we show that barley stripe mosaic virus (BSMV) infection activates the SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. The overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologs in Arabidopsis (Arabidopsis thaliana), NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity, thereby impairing downstream SA defense gene expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against lychnis ringspot virus, beet black scorch virus, and beet necrotic yellow vein virus. Taken together, our results reveal a role for the multifunctional γb protein in counteracting plant defense responses and an expanded broad-spectrum antibiotic role of the SA signaling pathway.

Disulfide reductase activity of thioredoxin-h2 imparts cold tolerance in Arabidopsis.

Many thioredoxin-h (Trx-h) proteins, cytosolic isotypes of Trxs, have been functionally characterized in plants; however, the physiological function of Arabidopsis Trx-h2, which harbors two active site cysteine (Cys) residues and an N-terminal extension peptide containing a fatty acid acylation site, remains unclear. In this study, we investigated the physiological function of Trx-h2 by performing several abiotic stress treatments using trx-h1-3 knockout mutant lines, and found that the reductase function of Trx-h2 is critical for cold resistance in Arabidopsis. Plants overexpressing Trx-h2 in the trx-h2 mutant background (Trx-h2OE/trx-h2) showed strong cold tolerant phenotypes compared with Col-0 (wild type) and trx-h2 mutant plants. By contrast, Trx-h2(C/S)OE/trx-h2 plants expressing a variant Trx-h2 protein, in which both active site Cys residues were substituted by serine (Ser) residues, showed high cold sensitivity, similar to trx-h2 plants. Moreover, cold-responsive (COR) genes were highly up-regulated in Trx-h2OE/trx-h2 plants but not in trx-h2 and Trx-h2(C/S)OE/trx-h2 plants under cold conditions. These results explicitly suggest that the cytosolic Trx-h2 protein relays the external cold stress signal to downstream cold defense signaling cascades through its protein disulfide reductase function.

Crystal structure of sulfonic peroxiredoxin Ahp1 in complex with thioredoxin Trx2 mimics a conformational intermediate during the catalytic cycle.

Peroxiredoxin (Prx) is a thiol-based peroxidase that eliminates reactive oxygen species to avoid oxidative damage. Alkyl hydroperoxide reductase Ahp1 is a novel and specific typical 2-cysteine Prx. Here, we present the crystal structure of sulfonic Ahp1 complexed with thioredoxin Trx2 at 2.12 Å resolution. This structure implies that the transient Ahp1-Trx2 complex during the catalytic cycle already have an ability to decompose the peroxides. Structural analysis reveals that the segment glutamine23-lysine32 juxtaposed to the resolving cysteine (CR) of Ahp1 moves inward to generate a compact structure upon peroxidatic cysteine (CP) overoxidation, resulting in the breakdown of several conserved hydrogen bonds formed by Ahp1-Trx2 complex interaction. Structural comparisons suggest that the structure of sulfonic Ahp1 represents a novel conformation of Ahp1, which can mimic a conformational intermediate between the reduced and oxidized forms. Therefore, this study may provide a new structural insight into the intermediate state in which the segment glutamine23-lysine32 juxtaposed to the cysteine31 (CR) undergoes a conformational change upon cysteine62 (CP) oxidation to prepare for the formation of an intermolecular CP-CR disulfide bond during Ahp1 catalytic cycle.

Overexpression of Tamarix hispida ThTrx5 Confers Salt Tolerance to Arabidopsis by Activating Stress Response Signals.

Salt stress inhibits normal plant growth and development by disrupting cellular water absorption and metabolism. Therefore, understanding plant salt tolerance mechanisms should provide a theoretical basis for developing salt-resistant varieties. Here, we cloned ThTrx5 from Tamarix hispida, a salt-resistant woody shrub, and generated ThTrx5-overexpressing transgenic Arabidopsis thaliana lines. Under NaCl stress, the germination rate of overexpressing ThTrx5 lines was significantly increased relative to that of the nontransgenic line; under salt stress, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione levels and root length and fresh weight values of transgenic ThTrx5 plants were significantly greater than corresponding values for wild-type plants. Moreover, with regard to the transcriptome, comparison of differential gene expression of transgenic versus nontransgenic lines at 0 h and 3 h of salt stress exposure revealed 500 and 194 differentially expressed genes (DEGs), respectively, that were mainly functionally linked to catalytic activity and binding process. Pull-down experiments showed that ThTrx bound 2-Cys peroxiredoxin BAS1-like protein that influences stress response-associated redox, hormone signal transduction, and transcription factor functions. Therefore, this work provides important insights into ThTrx5 mechanisms that promote salt tolerance in plants.

Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism.

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O2 conditions. However, an increase in the stoichiometry of photorespiratory CO2 release was found during O2 -dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD+ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.

Redox-mediated kick-start of mitochondrial energy metabolism drives resource-efficient seed germination.

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

Thioredoxin H (TrxH) contributes to adversity adaptation and pathogenicity of Edwardsiella piscicida.

Thioredoxins (Trxs) play an important role in defending against oxidative stress and keeping disulfide bonding correct to maintain protein function. Edwardsiella piscicida, a severe fish pathogen, has been shown to encode several thioredoxins including TrxA, TrxC, and TrxH, but their biological roles remain unknown. In this study, we characterized TrxH of E. piscicida (named TrxHEp) and examined its expression and function. TrxHEp is composed of 125 residues and possesses typical thioredoxin H motifs. Expression of trxHEp was upregulated under conditions of oxidative stress, iron starvation, low pH, and during infection of host cells. trxHEp expression was also regulated by ferric uptake regulator (Fur), an important global regulatory of E. piscicida. Compared to the wild type TX01, a markerless trxHEp in-frame mutant strain TX01∆trxH exhibited markedly compromised tolerance of the pathogen to hydrogen peroxide, acid stress, and iron deficiency. Deletion of trxHEp significantly retarded bacterial biofilm growth and decreased resistance against serum killing. Pathogenicity analysis shows that the inactivation of trxHEp significantly impaired the ability of E. piscicida to invade host cells, reproduce in macrophages, and infect host tissues. Introduction of a trans-expressed trxH gene restored the lost virulence of TX01∆trxH. There is likely to be a complex relationship of functional complementation or expression regulation between TrxH and another two thioredoxins, TrxA and TrxC, of E. piscicida. This is the first functional report of TrxH in fish pathogens, and the findings suggest that TrxHEp is essential for coping with adverse circumstances and contributes to host infection of E. piscicida.

Isolation and functional characterization of two thioredoxin h isoforms from grape.

Thioredoxins (Trxs) are small ubiquitous proteins that participate in dithiol-disulfide exchange reactions. In contrast to animals and prokaryotes, plants possess different types of Trxs that play a vital role in a number of different cellular processes. Two full-length cDNAs encoding different Trx h isoforms, designated VvTrx h2 and VvTrx h3, were isolated and cloned from grape (Vitis vinifera L. cv. Askari) berry tissue by rapid amplification of cDNA ends (RACE) method. VvTrx h2 and VvTrx h3 were heterologously expressed in Escherichia coli and their activities were compared using DTT-dependent insulin reduction and 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB) reduction activities. The NADPH-dependent DTNB reduction assay demonstrated that the both VvTrx h isoforms were reduced by NADPH-dependent thioredoxin reductase (NTR) from E. coli. Under heat shock treatment, the recombinant VvTrx h proteins formed the oligomeric structures at above 50 °C with a decrease in their disulfide reductase activities. The redox-dependent structural changes of VvTrx h2 and VvTrx h3 revealed that their oligomeric structures were changed into monomers and significantly increased their disulfide reductase activities. Furthermore, the both recombinant proteins were able to conserve a DTNB reduction activity even after 15 min heating at 99 °C.

Helicoverpa-inducible Thioredoxin h from Cicer arietinum: structural modeling and potential targets.

Thioredoxins are small and universal proteins, which are involved in the cell redox regulation. In plants, they participate in a broad range of biochemical processes like self-incompatibility, seed germination, pathogen & pest defense and oxidative stress tolerance. The h-type of thioredoxin (Trx-h) protein represents the largest Trx family. Herein, we characterized the Helicoverpa - inducible Trx h from an important legume, Cicer arietinum, CaHaTrx-h, 'CGFS' type Trxs, which encodes for a 113 amino acids long protein and possess characteristic motifs "FLKVDVDE" and "VVDFTASWCGPCRFIAPIL" and 73% sequence identity with AtTrx-h. Homology modeling and simulation of the target showed that the extended ß-sheet regions remain stable during the simulation while the helical regions fluctuate between alpha and 3-10 helical forms and highlights the flexibility of helix2-helix3 and terminal regions probably to accommodate an approaching protein target and facilitate their interaction. During the simulation, the structure exists in five energy minima clusters with biggest cluster size belonging to 20-25 ns time frames. PR-5 and Mannitol Dehydrogenase were nominated as potential targets and share close interaction with CaHaTrx-h via disulfide bond reduction. The study is an effort in the direction of understanding stress-related mechanisms in crop plants to overcome losses in agricultural yield.

The Path to Thioredoxin and Redox Regulation Beyond Chloroplasts.

Once the ferredoxin/thioredoxin system was established as a mechanism linking light to the post-translational regulation of chloroplast enzymes, I considered that plants might harbor a light-independent mechanism utilizing this same enzyme chemistry based on thiol-disulfide redox transitions. After reflection, it occurred to me that such a mechanism could be fundamental to seeds of cereals that undergo dramatic change following exposure to oxygen during maturation and drying. The pursuit of this idea led to the discovery of a family of extraplastidic thioredoxins, designated the h-type, that resemble animal and bacterial counterparts in undergoing enzymatic reduction with NADPH. Current evidence suggests that h-type thioredoxins function broadly throughout the plant. Here I describe how the thioredoxin h field developed, its current status and potential for contributing material benefits to society.

Two h-Type Thioredoxins Interact with the E2 Ubiquitin Conjugase PHO2 to Fine-Tune Phosphate Homeostasis in Rice.

Phosphate overaccumulator2 (PHO2) encodes a ubiquitin-conjugating E2 enzyme that is a major negative regulator of the inorganic phosphate (Pi)-starvation response-signaling pathway. A yeast two-hybrid (Y2H) screen in rice (Oryza sativa; Os) using OsPHO2 as bait revealed an interaction between OsPHO2 and two h-type thioredoxins, OsTrxh1 and OsTrxh4. These interactions were confirmed in vivo using bimolecular fluorescence complementation (BiFC) of OsPHO2 and OsTrxh1/h4 in rice protoplasts and by in vitro pull-down assays with 6His-tagged OsTrxh1/h4 and GST-tagged OsPHO2. Y2H assays revealed that amino acid Cys-445 of OsPHO2 and an N-terminal Cys in the "WCGPC" motif of Trxhs were required for the interaction. Split-ubiquitin Y2H analyses and BiFC assays in rice protoplasts confirmed the interaction of OsPHO2 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (OsPHF1), and PHOSPHATE1;2 (OsPHO1;2) in the endoplasmic reticulum and Golgi membrane system, where OsPHO2 mediates the degradation of OsPHF1 in both tobacco (Nicotiana benthamiana) leaves and rice seedlings. Characterization of rice pho2 complemented lines, transformed with an endogenous genomic OsPHO2 or OsPHO2C445S (a constitutively reduced form) fragment, indicated that OsPHO2C445S restored Pi concentration in rice to statistically significant lower levels compared to native OsPHO2 Moreover, the suppression of OsTrxh1 (knockdown and knockout) resulted in slightly higher Pi concentration than that of wild-type Nipponbare in leaves. These results demonstrate that OsPHO2 is under redox control by thioredoxins, which fine-tune its activity and link Pi homeostasis with redox balance in rice.

Seed thioredoxin h.

Thioredoxins are nearly ubiquitous disulfide reductases involved in a wide range of biochemical pathways in various biological systems, and also implicated in numerous biotechnological applications. Plants uniquely synthesize an array of thioredoxins targeted to different cell compartments, for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses on occurrence, reaction mechanisms, specificity, target protein identification, three-dimensional structure and various applications. The aim is to provide a general background as well as an update covering the most recent findings. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock.

Thioredoxin h isoforms from rice are differentially reduced by NADPH/thioredoxin or GSH/glutaredoxin systems.

Rice (Oryza sativa L.) has multiple potential genes encoding thioredoxin (Trx) h and NADP-thioredoxin reductase (NTR). These NTR and Trx h isoforms, known as cytoplasmic NTR/Trx system along with multiple members of glutaredoxin (Grx) family constitute a complex redox control system in rice. In the present study, we investigated the kinetic parameters of two rice NTRs, OsNTRA and OsNTRB, toward three endogenous Trx h isoforms, OsTrx1, OsTrx20, and OsTrx23. The results showed that in contrast with OsTrx1 and OsTrx23, the isoform OsTrx20 was not reduced by OsNTR isoforms. The kcat/Km values of OsNTRB and OsNTRA toward OsTrx1 was six- and 13-fold higher than those values toward OsTrx23, respectively, suggesting that OsNTR isoforms do not reduce different OsTrx h isoforms, equivalently. Furthermore, the possible reduction of OsTrx isoforms by the glutathione (GSH)/Grx system was investigated through the heterologous expression of a gene encoding OsGrx9, a bicysteinic CPYC Grx found in rice. Whereas OsTrx23 was not reduced by GSH, OsTrx20 and with less efficiently OsTrx1 were reduced by GSH or GSH/Grx. Therefore, it seems that OsTrx1 can be reduced either by OsNTR or GSH/Grx. These data for the first time provides an evidence for cross-talking between NTR/Trx and GSH/Grx systems in rice.

Mitogen-activated protein kinase 6 regulates NPR1 gene expression and activation during leaf senescence induced by salicylic acid.

Plant senescence is a highly regulated process that can be induced by a range of factors. The nonexpressor of pathogenesis-related genes 1 (npr1) mutant is defective in the salicylic acid (SA) signalling pathway, displaying delayed yellowing during developmental senescence. However, the regulating mechanism of NPR1 on exogenous SA-induced senescence in detached Arabidopsis leaves has not yet been clarified. It was shown here that mitogen-activated protein kinase 6 (MPK6) is involved in promoting exogenous SA-induced detached leaf senescence. During the process of SA-induced senescence, the expression of NPR1 and senescence-related transcription factor WRKY6 was suppressed in mpk6 mutant plants. Further analyses showed that the NPR1 mRNA level is reduced in wrky6 mutants and enhanced in WRKY6 overexpressing lines. Meanwhile, chromatin immunoprecipitation experiments revealed that WRKY6 binds directly to the NPR1 promoter containing W-box motifs. Moreover, inhibition of MPK6 function diminished SA-induced monomerization and nuclear localization of NPR1. In addition, the expression of Trx h5, which catalyses the SA-induced NPR1 activation, was suppressed in the mpk6 mutant, suggesting that MPK6 promotes NPR1 activation, possibly by regulating the expression of Trx h5. Collectively, MPK6-mediated WRKY6 and Trx h5 transcriptional activation co-regulated the expression of the NPR1 gene and the monomerization of NPR1 protein, allowing it to enter the nucleus, thereby promoting SA-induced leaf senescence. These results provide new insight into the mechanism of exogenous SA-induced detached leaf senescence.

Selective protein denitrosylation activity of Thioredoxin-h5 modulates plant Immunity.

In eukaryotes, bursts of reactive oxygen and nitrogen species mediate cellular responses to the environment by modifying cysteines of signaling proteins. Cysteine reactivity toward nitric oxide (NO) leads to formation of S-nitrosothiols (SNOs) that play important roles in pathogenesis and immunity. However, it remains poorly understood how SNOs are employed as specific, reversible signaling cues. Here we show that in plant immunity the oxidoreductase Thioredoxin-h5 (TRXh5) reverses SNO modifications by acting as a selective protein-SNO reductase. While TRXh5 failed to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, it rescued immunity in nox1 mutants that exhibit elevated levels of free NO. Rescue by TRXh5 was conferred through selective denitrosylation of excessive protein-SNO, which reinstated signaling by the immune hormone salicylic acid. Our data indicate that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signaling in plant immunity.

Glucitol dehydrogenase from peach (Prunus persica) fruits is regulated by thioredoxin h.

Glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. Herein we report the molecular cloning, heterologous expression and characterization of Gol dehydrogenase (GolDHase, EC 1.1.1.14) from peach (Prunus persica) fruits. The recombinant enzyme showed kinetic parameters similar to those reported for orthologous enzymes purified from apple and pear fruits. The activity of recombinant GolDHase was strongly inhibited by Cu(2+) and Hg(2+), suggesting that it might have cysteine residues critical for functionality. Oxidizing compounds (such as diamide, hydrogen peroxide and oxidized glutathione) inactivated the enzyme, whereas its activity was restored after incubation with reduced glutathione and thioredoxin from Escherichia coli. Recombinant thioredoxin h from peach fruits also recovered the activity of oxidized GolDHase. Our results suggest that peach fruit GolDHase could be redox regulated in vivo and this would be of relevance to determine carbon assimilation and partitioning in plants accumulating sugar alcohols.

Cloning and characterization of thioredoxin h in the three-pistil line of common wheat.

Thioredoxin h (Trxh) is a ubiquitous protein that reduces disulfides in target proteins, and is itself reduced by NADPH-thioredoxin reductase. In the current study, the complementary DNA sequence and the genomic sequence of the three-pistil (TP) line of common wheat (Triticum aestivum L.) were obtained from spikes through reverse transcription-polymerase chain reaction (RT-PCR) and touchdown-PCR. Sequence alignment of amino acids of TPTrxh then allowed for predictions of its physicochemical properties, secondary structures, tertiary structures, and functional domains. Furthermore, the TPTrxh gene was overexpressed in Escherichia coli and its activity was demonstrated using a dithiothreitol-dependent insulin assay. The expression patterns of TPTrxh were analyzed through real-time RT-PCR in different tissues and across different developmental stages of young spikes. The complementary DNA of TPTrxh was found to be 411 bp in length, encoding 118 amino acids. Its genomic sequence was determined to be 2632 bp, possessing 3 exons and 2 introns. Functional domain analysis indicated that TPTrxh contained a WCGPC motif located at the end of the second ß-fold and on the initial side of the second α-helix. The TPTrxh protein reduces intramolecular and intermolecular disulfide bridges in target proteins. Young spikes contain higher levels of TPTrxh transcripts than do stems and leaves. The transcript levels in the young spikes (2-5 mm in length) of the Chinese Spring TP line increased 2.84-fold relative to those of young spikes (2-5 mm in length) of the Chinese Spring line. These data provide a basis for future research into the function of Trxh, and offer further insight into the molecular mechanism of the TP mutation in wheat.

The role of thioredoxin h in protein metabolism during wheat (Triticum aestivum L.) seed germination.

Thioredoxin h can regulate the redox environment in the cell and play an important role in the germination of cereals. In the present study, the thioredoxin s antisense transgenic wheat with down-regulation of thioredoxin h was used to study the role of thioredoxin h in protein metabolism during germination of wheat seeds, and to explore the mechanism of the thioredoxin s antisense transgenic wheat seeds having high resistance to pre-harvest sprouting. The qRT-PCR results showed that the expression of protein disulfide isomerase in the thioredoxin s antisense transgenic wheat was up-regulated, which induced easily forming glutenin macropolymers and the resistance of storage proteins to degradation. The expression of serine protease inhibitor was also up-regulated in transgenic wheat, which might be responsible for the decreased activity of thiocalsin during the germination. The expression of WRKY6 in transgenic wheat was down-regulated, which was consistent with the decreased activity of glutamine oxoglutarate aminotransferase. In transgenic wheat, the activities of glutamate dehydrogenase, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were down-regulated, indicating that the metabolism of amino acid was lower than that in wild-type wheat during seed germination. A putative model for the role of thioredoxin h in protein metabolism during wheat seed germination was proposed and discussed.

Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study.

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.