Molecular characterization and expression profiling of the protein disulfide isomerase gene family in Brachypodium distachyon L.
ABSTRACT: Protein disulfide isomerases (PDI) are involved in catalyzing protein disulfide bonding and isomerization in the endoplasmic reticulum and functions as a chaperone to inhibit the aggregation of misfolded proteins. Brachypodium distachyon is a widely used model plant for temperate grass species such as wheat and barley. In this work, we report the first molecular characterization, phylogenies, and expression profiles of PDI and PDI-like (PDIL) genes in B. distachyon in different tissues under various abiotic stresses. Eleven PDI and PDIL genes in the B. distachyon genome by in silico identification were evenly distributed across all five chromosomes. The plant PDI family has three conserved motifs that are involved in catalyzing protein disulfide bonding and isomerization, but a different exon/intron structural organization showed a high degree of structural differentiation. Two pairs of genes (BdPDIL4-1 and BdPDIL4-2; BdPDIL7-1 and BdPDIL7-2) contained segmental duplications, indicating each pair originated from one progenitor. Promoter analysis showed that Brachypodium PDI family members contained important cis-acting regulatory elements involved in seed storage protein synthesis and diverse stress response. All Brachypodium PDI genes investigated were ubiquitously expressed in different organs, but differentiation in expression levels among different genes and organs was clear. BdPDIL1-1 and BdPDIL5-1 were expressed abundantly in developing grains, suggesting that they have important roles in synthesis and accumulation of seed storage proteins. Diverse treatments (drought, salt, ABA, and H2O2) induced up- and down-regulated expression of Brachypodium PDI genes in seedling leaves. Interestingly, BdPDIL1-1 displayed significantly up-regulated expression following all abiotic stress treatments, indicating that it could be involved in multiple stress responses. Our results provide new insights into the structural and functional characteristics of the plant PDI gene family.
Project description:Protein disulfide isomerases (PDIs) are responsible for catalyzing the proper oxidation and isomerization of disulfide bonds of newly synthesized proteins in the endoplasmic reticulum (ER). Here, it is shown that human PDI (PDIA1) dimerizes in vivo and proposed that the dimerization of PDI has physiological relevance by autoregulating its activity. The crystal structure of the dimeric form of noncatalytic bb' domains of human PDIA1 determined to 2.3 Å resolution revealed that the formation of dimers occludes the substrate binding site and may function as a mechanism to regulate PDI activity in the ER.
Project description:Living organisms are constantly subject to DNA damage from environmental sources. Due to the sessile nature of plants, UV irradiation is a major genotoxic agent and imposes a significant threat on plant survival, genome stability and crop yield. In addition, other environmental chemicals can also influence the stability of the plant genome. Eukaryotic organisms have evolved a mechanism to cope with replication-blocking lesions and stabilize the genome. This mechanism is known as error-free DNA damage tolerance, and is mediated by K63-linked PCNA polyubiquitination. Genes related to K63-linked polyubiquitination have been isolated recently from model plants like Arabidopsis and rice, but we are unaware of such reports on the crop model Brachypodium distachyon. Here, we report the identification and functional characterization of two B. distachyon UBC13 genes. Both Ubc13s form heterodimers with Uevs from other species, which are capable of catalyzing K63 polyubiquitination in vitro. Both genes can functionally rescue the yeast ubc13 null mutant from killing by DNA-damaging agents. These results suggest that Ubc13-Uev-promoted K63-linked polyubiquitination is highly conserved in eukaryotes including B. distachyon. Consistent with recent findings that K63-linked polyubiquitination is involved in several developmental and stress-responsive pathways, the expression of BdUbc13s appears to be constitutive and is regulated by abnormal temperatures.
Project description:Redox modulation of cysteine residues is one of the post-translational modifications of N-methyl-D-aspartate receptor (NMDAR). Protein disulfide isomerases (PDI), an endoplasmic reticulum (ER) chaperone, plays a crucial role in catalyzing disulfide bond formation, reduction, and isomerization. In the present study, we found that PDI bound to NMDAR in the normal hippocampus, and that this binding was increased in chronic epileptic rats. In vitro thiol reductase assay revealed that PDI increased the amount of thiols on full-length recombinant NR1 protein. PDI siRNA, 5-5'-dithio-bis(2-nitrobenzoic acid) (DTNB), bacitracin and PDI antibody reduced seizure susceptibility in response to pilocarpine. In addition, PDI knockdown effectively ameliorated spontaneous seizure activity in chronic epileptic rats. Anticonvulsive effects of PDI siRNA were correlated to the reduction of the amount of free- and nitrosothiols on NMDAR, accompanied by the inhibition of PDI activity. However, PDI knockdown did not lead to alteration in basal neurotransmission or ER stress under physiological condition. These findings provide mechanistic insight into sulfhydration of disulfide bonds on NMDAR by PDI, and suggest that PDI may represent a target of potential therapeutics for epilepsy, which avoids a possible side effect on physiological receptor functionality.
Project description:About one-third of all cellular proteins pass through the secretory pathway and hence undergo oxidative folding in the endoplasmic reticulum (ER). Protein-disulfide isomerase (PDI) and related members of the PDI family assist in the folding of substrates by catalyzing the oxidation of two cysteines and isomerization of disulfide bonds as well as by acting as chaperones. In this study, we present the crystal structure of ERp27, a redox-inactive member of the PDI family. The structure reveals its substrate-binding cleft, which is homologous to PDI, but is able to adapt in size and hydrophobicity. Isothermal titration calorimetry experiments demonstrate that ERp27 is able to distinguish between folded and unfolded substrates, only interacting with the latter. ERp27 is up-regulated during ER stress, thus presumably allowing it to bind accumulating misfolded substrates and present them to ERp57 for catalysis.
Project description:MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKC(c)-type, 7 MIKC*-type, 9 M?, 7 M? and 2 M? MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.
Project description:BACKGROUND:Little is known about the potential of Brachypodium distachyon as a model for low temperature stress responses in Pooideae. The ice recrystallization inhibition protein (IRIP) genes, fructosyltransferase (FST) genes, and many C-repeat binding factor (CBF) genes are Pooideae specific and important in low temperature responses. Here we used comparative analyses to study conservation and evolution of these gene families in B. distachyon to better understand its potential as a model species for agriculturally important temperate grasses. RESULTS:Brachypodium distachyon contains cold responsive IRIP genes which have evolved through Brachypodium specific gene family expansions. A large cold responsive CBF3 subfamily was identified in B. distachyon, while CBF4 homologs are absent from the genome. No B. distachyon FST gene homologs encode typical core Pooideae FST-motifs and low temperature induced fructan accumulation was dramatically different in B. distachyon compared to core Pooideae species. CONCLUSIONS:We conclude that B. distachyon can serve as an interesting model for specific molecular mechanisms involved in low temperature responses in core Pooideae species. However, the evolutionary history of key genes involved in low temperature responses has been different in Brachypodium and core Pooideae species. These differences limit the use of B. distachyon as a model for holistic studies relevant for agricultural core Pooideae species.
Project description:Brachypodium distachyon is a new model plant closely related to wheat and other cereals. In this study, we performed a comprehensive analysis of hormone-regulated genes in Brachypodium distachyon using RNA sequencing technology. Brachypodium distachyon seedlings were treated with eight phytohormones (auxin, cytokinine, brassinosteroid, gibberelline, abscisic acid, ethylene, jasmonate and salicylic acid) and two inhibitors, Brz220 (brassinosteroid biosynthesis inhibitor) and prohexadione (gibberelline biosynthesis inhibitor). The expressions of 1807 genes were regulated in a phytohormone-dependent manner. We compared the data with the phytohormone responses that have reported in rice. Transcriptional responses to hormones are conserved between Bracypodium and rice. Transcriptional regulation by brassinosteroid, gibberellin and ethylene was relatively weaker than those by other hormones. This is consistent with the data obtained from comprehensive analysis of hormone responses reported in Arabidopsis. Brachypodium and Arabidopsis also shared some common transcriptional responses to phytohormones. Alternatively, unique transcriptional responses to phytohormones were observed in Brachypodium. For example, the expressions of ACC synthase genes were up-regulated by auxin treatment in rice and Arabidopsis, but no orthologous ACC synthase gene was up-regulated in Brachypodium. Our results provide information useful to understand the diversity and similarity of hormone-regulated transcriptional responses between eudicots and monocots.
Project description:In the endoplasmic reticulum (ER), ER oxidoreductin 1 (ERO1) catalyzes intramolecular disulfide-bond formation within its substrates in coordination with protein-disulfide isomerase (PDI) and related enzymes. However, the molecular mechanisms that regulate the ERO1-PDI system in plants are unknown. Reduction of the regulatory disulfide bonds of the ERO1 from soybean, GmERO1a, is catalyzed by enzymes in five classes of PDI family proteins. Here, using recombinant proteins, vacuum-ultraviolet circular dichroism spectroscopy, biochemical and protein refolding assays, and quantitative immunoblotting, we found that GmERO1a activity is regulated by reduction of intramolecular disulfide bonds involving Cys-121 and Cys-146, which are located in a disordered region, similarly to their locations in human ERO1. Moreover, a GmERO1a variant in which Cys-121 and Cys-146 were replaced with Ala residues exhibited hyperactive oxidation. Soybean PDI family proteins differed in their ability to regulate GmERO1a. Unlike yeast and human ERO1s, for which PDI is the preferred substrate, GmERO1a directly transferred disulfide bonds to the specific active center of members of five classes of PDI family proteins. Of these proteins, GmPDIS-1, GmPDIS-2, GmPDIM, and GmPDIL7 (which are group II PDI family proteins) failed to catalyze effective oxidative folding of substrate RNase A when there was an unregulated supply of disulfide bonds from the C121A/C146A hyperactive mutant GmERO1a, because of its low disulfide-bond isomerization activity. We conclude that regulation of plant ERO1 activity is particularly important for effective oxidative protein folding by group II PDI family proteins.
Project description:Protein disulfide isomerase (PDI) and PDI-like proteins contain thioredoxin domains that catalyze protein disulfide bond, inhibit aggregation of misfolded proteins, and function in isomerization during protein folding in endoplasmic reticulum and responses during abiotic stresses.Chinese cabbage is widely recognized as an economically important, nutritious vegetable, but its yield is severely hampered by various biotic and abiotic stresses. Because of, it is prime need to identify those genes whose are responsible for biotic and abiotic stress tolerance. PDI family genes are among of them.We have identified 32 PDI genes from the Br135K microarray dataset, NCBI and BRAD database, and in silico characterized their sequences. Expression profiling of those genes was performed using cDNA of plant samples imposed to abiotic stresses; cold, salt, drought and ABA (Abscisic Acid) and biotic stress; Fusarium oxysporum f. sp. conglutinans infection. The Chinese cabbage PDI genes were clustered in eleven groups in phylogeny. Among them, 15 PDI genes were ubiquitously expressed in various organs, while 24 PDI genes were up-regulated under salt and drought stress. By contrast, cold and ABA stress responsive gene number were ten and nine, respectively. In case of F. oxysporum f. sp. conglutinans infection 14 BrPDI genes were highly up-regulated. Interestingly, BrPDI1-1 gene was identified as putative candidate against abiotic (salt and drought) and biotic stresses, BrPDI5-2 gene for ABA stress, and BrPDI1-4, 6-1 and 9-2 were putative candidate genes for both cold and chilling injury stresses.Our findings help to elucidate the involvement of PDI genes in stress responses, and they lay the foundation for functional genomics in future studies and molecular breeding of Brassica rapa crops. The stress-responsive PDI genes could be potential resources for molecular breeding of Brassica crops resistant to biotic and abiotic stresses.
Project description:Protein disulfide isomerases (PDI) and PDI-like proteins catalyze the formation and isomerization of protein disulfide bonds in the endoplasmic reticulum and prevent the buildup of misfolded proteins under abiotic stress conditions. In the present study, we conducted the first comprehensive genome-wide exploration of the <i>PDI</i> gene family in tomato (<i>Solanum lycopersicum</i> L.). We identified 19 tomato <i>PDI</i> genes that were unevenly distributed on 8 of the 12 tomato chromosomes, with segmental duplications detected for 3 paralogous gene pairs. Expression profiling of the <i>PDI</i> genes revealed that most of them were differentially expressed across different organs and developmental stages of the fruit. Furthermore, most of the <i>PDI</i> genes were highly induced by heat, salt, and abscisic acid (ABA) treatments, while relatively few of the genes were induced by cold and nutrient and water deficit (NWD) stresses. The predominant expression of <i>SlPDI1-1</i>, <i>SlPDI1-3</i>, <i>SlPDI1-4</i>, <i>SlPDI2-1</i>, <i>SlPDI4-1</i>, and <i>SlPDI5-1</i> in response to abiotic stress and ABA treatment suggested they play regulatory roles in abiotic stress tolerance in tomato in an ABA-dependent manner. Our results provide new insight into the structure and function of <i>PDI</i> genes and will be helpful for the selection of candidate genes involved in fruit development and abiotic stress tolerance in tomato.