HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis.

Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box-like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

Overexpression of rice F-box protein OsFBX322 confers increased sensitivity to gamma irradiation in Arabidopsis.

Ionizing radiation has a substantial effect on physiological and biochemical processes in plants via induction of transcriptional changes and diverse genetic alterations. Previous microarray analysis showed that rice OsFBX322, which encodes a rice F-box protein, was downregulated in response to three types of ionizing radiation: gamma irradiation, ion beams, and cosmic rays. In order to characterize the radiation-responsive genes in rice, OsFBX322 was selected for further analysis. OsFBX322 expression patterns in response to radiation were confirmed using quantitative RT-PCR. Transient expression of a GFP-OsFBX322 fusion protein in tobacco leaf epidermis indicated that OsFBX322 is localized to the nucleus. To determine the effect of OsFBX322 expression on radiation response, OsFBX322 was overexpressed in Arabidopsis. Transgenic overexpression lines were more sensitive to gamma irradiation than control plants. These results suggest that OsFBX322 plays a negative role in the defense response to radiation in plants. In addition, we obtained four co-expression genes of OsFBX322 by specific co-expression networks using the ARANCE. Quantitative RT-PCR showed that the four genes were also downregulated after exposure to the three types of radiation. These results imply that the co-expressed genes may serve as key regulators in the radiation response pathway in plants.

Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis.

BACKGROUND: Jacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation. RESULTS: Time- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11. CONCLUSIONS: OsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage.

Phytochrome B Positively Regulates Red Light-Mediated ER Stress Response in Arabidopsis.

Light plays a crucial role in plant growth and development, and light signaling is integrated with various stress responses to adapt to different environmental changes. During this process, excessive protein synthesis overwhelms the protein-folding ability of the endoplasmic reticulum (ER), causing ER stress. Although crosstalk between light signaling and ER stress response has been reported in plants, the molecular mechanisms underlying this crosstalk are poorly understood. Here, we demonstrate that the photoreceptor phytochrome B (phyB) induces the expression of ER luminal protein chaperones as well as that of unfolded protein response (UPR) genes. The phyB-5 mutant was less sensitive to tunicamycin (TM)-induced ER stress than were the wild-type plants, whereas phyB-overexpressing plants displayed a more sensitive phenotype under white light conditions. ER stress response genes (BiP2 and BiP3), UPR-related bZIP transcription factors (bZIP17, bZIP28, and bZIP60), and programmed cell death (PCD)-associated genes (OXI1, NRP1, and MC8) were upregulated in phyB-overexpressing plants, but not in phyB-5, under ER stress conditions. The ER stress-sensitive phenotype of phyB-5 under red light conditions was eliminated with a reduction in photo-equilibrium by far-red light and darkness. The N-terminal domain of phyB is essential for signal transduction of the ER stress response in the nucleus, which is similar to light signaling. Taken together, our results suggest that phyB integrates light signaling with the UPR to relieve ER stress and maintain proper plant growth.

Proteomic identification of an embryo-specific 1Cys-Prx promoter and analysis of its activity in transgenic rice.

Proteomic analysis of a rice callus led to the identification of 10 abscisic acid (ABA)-induced proteins as putative products of the embryo-specific promoter candidates. 5'-flanking sequence of 1 Cys-Prx, a highly-induced protein gene, was cloned and analyzed. The transcription initiation site of 1 Cys-Prx maps 96 nucleotides upstream of the translation initiation codon and a TATA-box and putative seed-specific cis-acting elements, RYE and ABRE, are located 26, 115 and 124 bp upstream of the transcription site, respectively. ß-glucuronidase (GUS) expression driven by the 1 Cys-Prx promoters was strong in the embryo and aleurone layer and the activity reached up to 24.9 ± 3.3 and 40.5 ± 2.1 pmol (4 MU/min/µg protein) in transgenic rice seeds and calluses, respectively. The activity of the 1 Cys-Prx promoters is much higher than that of the previously-identified embryo-specific promoters, and comparable to that of strong endosperm-specific promoters in rice. GUS expression driven by the 1 Cys-Prx promoters has been increased by ABA treatment and rapidly induced by wounding in callus and at the leaf of the transgenic plants, respectively. Furthermore, ectopic expression of the GUS construct in Arabidopsis suggested that the 1 Cys-Prx promoter also has strong activity in seeds of dicot plants.

ZIP8 exacerbates collagen-induced arthritis by increasing pathogenic T cell responses.

Zinc is a trace element that is essential for immune responses. Therefore, changes in cellular zinc levels in specific immune cells may influence inflammatory autoimmune diseases, such as rheumatoid arthritis (RA). However, the regulation of zinc mobilization in immune cells and its role in the pathogenesis of RA are not fully understood. Thus, we investigated the roles of zinc transporters in RA pathogenesis. We demonstrated that ZIP8 was specifically upregulated in CD4+ T cells that infiltrated the inflamed joint and that ZIP8 deficiency in CD4+ T cells abrogated collagen-induced arthritis. ZIP8 deficiency dramatically affected zinc influx in effector T cells and profoundly reduced T cell receptor (TCR)-mediated signaling, including NF-κB and MAPK signaling, which are pathways that are involved in T helper (Th) 17 cell differentiation. Taken together, our findings suggest that ZIP8 depletion in CD4+ T cells attenuates TCR signaling due to insufficient cellular zinc, thereby reducing the function of effector CD4+ T cells, including Th17 cells. Our results also suggest that targeting ZIP8 may be a useful strategy to inhibit RA development and pathogenesis.

MiR-921 directly downregulates GPx3 in A549 lung cancer cells.

Glutathione peroxidase 3 (GPx3), a major antioxidant enzyme in plasma, catalyzes the reduction of H2O2, lipid peroxides and organic hydroperoxides by reducing glutathione (GSH). Hypermethylation of the GPx3 promoter and suppression of GPx3 expression are associated with inflammation, tumorigenesis, and response to chemotherapy in various types of cancer. We previously reported the possibility of GPx3 as a serological marker for lung cancer. In this study, we assessed the role of the microRNA (miRNA) hsa-miR-921 (miR-921) in the regulation of GPx3 expression in A549 lung cancer cells. The expression patterns of the miRNAs of A549, H1650, and H1975 cells were compared and analyzed. Of 25 miRNAs from the A549 cell line, the expression of 10 decreased and the expression of 15 increased in comparison to the miRNAs from the other cell lines. Of the miRNAs with reduced expression, the most reduced miRNA was miR-921 and the expected binding site of which is in the 3'-untranslated region (UTR) of GPx3. We found that miR-921 inhibited the expression of GPx3 and bound directly to the 3'-UTR of GPx3.

Molecular and functional characterization of monocot-specific Pex5p splicing variants, using OsPex5pL and OsPex5pS from rice (Oryza sativa).

We identified two alternatively spliced variants of the peroxisomal targeting signal 1 (PTS1) receptor protein Pex5ps in monocot (rice, wheat, and barley) but not in dicot (Arabidopsis and tobacco) plants. We characterized the molecular and functional differences between the rice (Oryza sativa) Pex5 splicing variants OsPex5pL and OsPex5pS. There is only a single-copy of OsPEX5 in the rice genome and RT-PCR analysis points to alternative splicing of the transcripts. Putative light-responsive cis-elements were identified in the 5' region flanking OsPEX5L and Northern blot analysis demonstrated that this region affected light-dependent expression of OsPEX5 transcription. Using the pex5-deficient yeast mutant Scpex5, we showed that OsPex5pL and OsPex5pS are able to restore translocation of a model PTS1 protein (GFP-SKL) into peroxisomes. OsPex5pL and OsPex5pS formed homo-complexes via specific interaction domains, and interacted with each other and OsPex14p to form hetero-complexes. Although overexpression of OsPex5pL in the Arabidopsis pex5 mutant (Atpex5) rescued the mutant phenotype, overexpression of OsPex5pS only resulted in partial recovery.

Molecular dissection of the response of the rice Systemic Acquired Resistance Deficient 1 (SARD1) gene to different types of ionizing radiation.

PURPOSE: Exposure to ionizing radiation induces plant defenses by regulating the expression of response genes. The systemic acquired resistance deficient 1 (SARD1) is a key gene in plant defense response. In this study, the function of Oryza sativa SARD1 (OsSARD1) was investigated after exposure of seeds/plants to ionizing radiation, jasmonic acid (JA) or salicylic acid (SA). MATERIALS AND METHODS: Rice seeds exposed to two types of ionizing radiations (gamma ray [GR] and ion beam [IB]) were analyzed by quantitative reverse transcription PCR (qRT-PCR) to identify the genes that are altered in response to ionizing radiation. Then, OsSARD1-overexpressing homozygous Arabidopsis plants were generated to assess the effects of OsSARD1 in the response to irradiation. The phenotypes of these transgenic plants, as well as control plants, were monitored after GR irradiation at doses of 200 and 300 Gray (Gy). RESULTS: The OsSARD1 transcript was strongly downregulated after exposure to GR and IB irradiation. Previous phylogenetic analysis showed that the Arabidopsis SARD1 (AtSARD1) protein is closely related to Arabidopsis calmodulin-binding protein 60g (AtCBP60g), which is known to be required for activation of SA biosynthesis. In this study, phylogenetic analysis showed that OsSARD1 was grouped with AtSARD1. The OsSARD1 gene was induced after exposure to SA and JA. The biological phenotype of OsSARD1-overexpressing Arabidopsis plants was examined. OsSARD1-overexpressing plants displayed resistance to GR; in comparison with wild-type plants, the height and weight of OsSARD1-overexpressing plants were significantly greater after GR irradiation. In addition, OsSARD1 protein was abundantly accumulated in the nucleus. CONCLUSIONS: The results indicate that OsSARD1 plays an important role in the regulation of the defense responses to GR and IB irradiation and exhibits phytohormone induced expression.

A novel thiol-reductase activity of Arabidopsis YUC6 confers drought tolerance independently of auxin biosynthesis.

YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses.

NADPH-dependent thioredoxin reductase A (NTRA) confers elevated tolerance to oxidative stress and drought.

NADPH-dependent thioredoxin reductases (NTRs) are key-regulatory enzymes determining the redox state of the thioredoxin (Trx) system that provides reducing power to peroxidases or oxidoreductases. Moreover, it also plays an essential function in the direct reduction of ROS and acquiring stress tolerance in plant. Cytoplasmic NTRA, mitochondrial NTRB, and chloroplastic NTRC are the three conserved NTRs which cooperate with specific sub-cellularly localized Trxs in Arabidopsis. However, cytosolic NTRs such as NTRA in Arabidopsis have not previously been identified in plants or mammals as a source of functional redundancy with mitochondrial NTRs. Here, we show the involvement of NTRA in the plant stress response counteracting oxidative and drought stresses. Methyl viologen (MV), an inducer of oxidative stress in plants, enhanced the NTRA transcripts. To identify the physiological role of NTRA influencing ROS homeostasis by stress, NTRA overexpression (NTRAOX) and knock-out mutants (ntra-ko) were generated. After exposure to oxidative stress, wild-type and ntra-ko plants were sensitive, but NTRAOX plants tolerant. ROS range was increased by MV in wild-type and ntra-ko plants, but not in NTRAOX. Investigating the involvement of Arabidopsis NTRA in drought, NTRAOX plants exhibited extreme drought tolerance with high survival rates, lower water loss and reduced ROS compared to wild-type and ntra-ko plants. Transcripts of drought-responsive genes, such as RD29A and DREB2A, were highly expressed under drought and antioxidant genes, namely CuZnSOD and APX1 were enhanced in the absence of drought in NTRAOX plants. The results suggest that NTRA overexpression confers oxidative and drought tolerance by regulation of ROS amounts.

Dual functions of Arabidopsis sulfiredoxin: acting as a redox-dependent sulfinic acid reductase and as a redox-independent nuclease enzyme.

Based on the fact that the amino acid sequence of sulfiredoxin (Srx), already known as a redox-dependent sulfinic acid reductase, showed a high sequence homology with that of ParB, a nuclease enzyme, we examined the nucleic acid binding and hydrolyzing activity of the recombinant Srx in Arabidopsis (AtSrx). We found that AtSrx functions as a nuclease enzyme that can use single-stranded and double-stranded DNAs as substrates. The nuclease activity was enhanced by divalent cations. Particularly, by point-mutating the active site of sulfinate reductase, Cys (72) to Ser (AtSrx-C72S), we demonstrate that the active site of the reductase function of AtSrx is not involved in its nuclease function.