Elongation of Siliques Without Pollination 3 Regulates Nutrient Flow Necessary for Embryogenesis.

Apomixis, defined as the transfer of maternal germplasm to offspring without fertilization, enables the fixation of F1-useful traits, providing advantages in crop breeding. However, most apomictic plants require pollination to produce the endosperm. The endosperm is essential for embryogenesis, and its development is suppressed until fertilization. We show that the expression of a chimeric repressor of the Elongation of Siliques without Pollination 3 (ESP3) gene (Pro35S:ESP3-SRDX) induces ovule enlargement without fertilization in Arabidopsis thaliana. The ESP3 gene encodes a protein similar to the flowering Wageningen homeodomain transcription factor containing a StAR-related lipid transfer domain. However, ESP3 lacks the homeobox-encoding region. Genes related to the cell cycle and sugar metabolism were upregulated in unfertilized Pro35S:ESP3-SRDX ovules similar to those in fertilized seeds, while those related to autophagy were downregulated similar to those in fertilized seeds. Unfertilized Pro35S:ESP3-SRDX ovules partially nourished embryos when only the egg was fertilized, accumulating hexoses without central cell proliferation. ESP3 may regulate nutrient flow during seed development, and ESP3-SRDX could be a useful tool for complete apomixis that does not require pseudo-fertilization.

The CIB1 transcription factor regulates light- and heat-inducible cell elongation via a two-step HLH/bHLH system.

Light and high temperature promote plant cell elongation. PHYTOCHROME INTERACTING FACTOR4 (PIF4, a typical basic helix-loop-helix [bHLH] transcriptional activator) and the non-DNA binding atypical HLH inhibitors PHYTOCHROME RAPIDLY REGULATED1 (PAR1) and LONG HYPOCOTYL IN FAR-RED 1 (HFR1) competitively regulate cell elongation in response to light conditions and high temperature. However, the underlying mechanisms have not been fully clarified. Here, we show that in Arabidopsis thaliana, the bHLH transcription factor CRYPTOCHROME-INTERACTING BASIC HELIX-LOOP-HELIX 1 (CIB1) positively regulates cell elongation under the control of PIF4, PAR1, and HFR1. Furthermore, PIF4 directly regulates CIB1 expression by interacting with its promoter, and PAR1 and HFR1 interfere with PIF4 binding to the CIB1 promoter. CIB1 activates genes that function in cell elongation, and PAR1 interferes with the DNA binding activity of CIB1, thus suppressing cell elongation. Hence, two antagonistic HLH/bHLH systems, the PIF4-PAR1/HFR1 and CIB1-PAR1 systems, regulate cell elongation in response to light and high temperature. We thus demonstrate the important role of non-DNA binding small HLH proteins in the transcriptional regulation of cell elongation in plants.

Arabidopsis thaliana NGATHA1 transcription factor induces ABA biosynthesis by activating NCED3 gene during dehydration stress.

The plant hormone abscisic acid (ABA) is accumulated after drought stress and plays critical roles in the responses to drought stress in plants, such as gene regulation, stomatal closure, seed maturation, and dormancy. Although previous reports revealed detailed molecular roles of ABA in stress responses, the factors that contribute to the drought-stress responses-in particular, regulation of ABA accumulation-remain unclear. The enzyme NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3 (NCED3) is essential for ABA biosynthesis during drought stress, and the NCED3 gene is highly induced by drought stress. In the present study, we isolated NGATHAs (NGAs) as candidate transcriptional regulators of NCED3 through a screen of a plant library harboring the transcription factors fused to a chimeric repressor domain, SRDX. The NGA proteins were directly bound to a cis-element NGA-binding element (NBE) in the 5' untranslated region (5' UTR) of the NCED3 promoter and were suggested to be transcriptional activators of NCED3 Among the single-knockout mutants of four NGA family genes, we found that the NGATHA1 (NGA1) knockout mutant was drought-stress-sensitive with a decreased expression level of NCED3 during dehydration stress. These results suggested that NGA1 essentially functions as a transcriptional activator of NCED3 among the NGA family proteins. Moreover, the NGA1 protein was degraded under nonstressed conditions, and dehydration stress enhanced the accumulation of NGA1 proteins, even in ABA-deficient mutant plants, indicating that there should be ABA-independent posttranslational regulations. These findings emphasize the regulatory mechanisms of ABA biosynthesis during early drought stress.

ABA Suppresses Root Hair Growth via the OBP4 Transcriptional Regulator.

Plants modify organ growth and tune morphogenesis in response to various endogenous and environmental cues. At the cellular level, organ growth is often adjusted by alterations in cell growth, but the molecular mechanisms underlying this control remain poorly understood. In this study, we identify the DNA BINDING WITH ONE FINGER (DOF)-type transcription regulator OBF BINDING PROTEIN4 (OBP4) as a repressor of cell growth. Ectopic expression of OBP4 in Arabidopsis (Arabidopsis thaliana) inhibits cell growth, resulting in severe dwarfism and the repression of genes involved in the regulation of water transport, root hair development, and stress responses. Among the basic helix-loop-helix transcription factors known to control root hair growth, OBP4 binds the ROOT HAIR DEFECTIVE6-LIKE2 (RSL2) promoter to repress its expression. The accumulation of OBP4 proteins is detected in expanding root epidermal cells, and its expression level is increased by the application of abscisic acid (ABA) at concentrations sufficient to inhibit root hair growth. ABA-dependent induction of OBP4 is associated with the reduced expression of RSL2 Furthermore, ectopic expression of OBP4 or loss of RSL2 function results in ABA-insensitive root hair growth. Taken together, our results suggest that OBP4-mediated transcriptional repression of RSL2 contributes to the ABA-dependent inhibition of root hair growth in Arabidopsis.

Decrease in Serum Amyloid a Protein Levels Following Three-month Stays in Negatively Charged Particle-dominant Indoor Air Conditions.

OBJECTIVE: The changes in serum adipokines and cytokines related to oxidative stress were examined during 3 months 'Off to On' and 'On to Off' periods using negatively charged particle-dominant indoor air conditions (NCPDIAC). METHODS: Seven volunteers participated in the study, which included 'OFF to 3 months ON' periods (ON trials) for a total of 16 times, and 'ON to 3 months OFF' (OFF trials) periods for a total of 13 times. RESULTS: With the exception of one case, serum amyloid A (SAA) levels decreased significantly during the ON trials. CONCLUSION: Considering that SAA is an acute phase reactive protein such as C reactive protein (CRP), this observed decrease might indicate the prevention of cardiovascular and atherosclerotic changes, since an increase in high-sensitive CRP is associated with the subsequent detection of these events.

Search for biomarkers of asbestos exposure and asbestos-induced cancers in investigations of the immunological effects of asbestos.

The immunological effects of asbestos exposure on various lymphocytes such as the regulatory T cell (Treg), responder CD4+ T helper cell (Tresp), CD8+ cytotoxic T lymphocytes (CTL), and natural killer (NK) cells were investigated. Results show that asbestos exposure impairs antitumor immunity through enhancement of regulatory T cell function and volume, reduction of CXCR3 chemokine receptor in responder CD4+ T helper cells, and impairment of the killing activities of CD8+ cytotoxic T lymphocytes (CTL) and NK cells. These findings were used to explore biological markers associated with asbestos exposure and asbestos-induced cancers and suggested the usefulness of serum/plasma IL-10 and TGF-ß, surface CXCR3 expression in Tresp, the secreting potential of IFN-γ in Tresp, intracellular perforin level in CTL, and surface expression NKp46 in NK cells. Although other unexplored cytokines in serum/plasma and molecules in these immunological cells, including Th17, should be investigated by experimental procedures in addition to a comprehensive analysis of screening methods, biomarkers based on immunological alterations may be helpful in clinical situations to screen the high-risk population exposed to asbestos and susceptible to asbestos-related cancers such as mesothelioma.

Induction of a dwarf phenotype with IBH1 may enable increased production of plant-made pharmaceuticals in plant factory conditions.

Year-round production in a contained, environmentally controlled 'plant factory' may provide a cost-effective method to produce pharmaceuticals and other high-value products. However, cost-effective production may require substantial modification of the host plant phenotype; for example, using dwarf plants can enable the growth of more plants in a given volume by allowing more plants per shelf and enabling more shelves to be stacked vertically. We show here that the expression of the chimeric repressor for Arabidopsis AtIBH1 (P35S:AtIBH1SRDX) in transgenic tobacco plants (Nicotiana tabacum) induces a dwarf phenotype, with reduced cell size. We estimate that, in a given volume of cultivation space, we can grow five times more AtIBH1SRDX plants than wild-type plants. Although, the AtIBH1SRDX plants also showed reduced biomass compared with wild-type plants, they produced about four times more biomass per unit of cultivation volume. To test whether the dwarf phenotype affects the production of recombinant proteins, we expressed the genes for anti-hepatitis B virus antibodies (anti-HBs) in tobacco plants and found that the production of anti-HBs per unit fresh weight did not significantly differ between wild-type and AtIBH1SRDX plants. These data indicate that P35S:AtIBH1SRDX plants produced about fourfold more antibody per unit of cultivation volume, compared with wild type. Our results indicate that AtIBH1SRDX provides a useful tool for the modification of plant phenotype for cost-effective production of high-value products by stably transformed plants in plant factory conditions.

A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis.

In plants, basic helix-loop-helix (bHLH) transcription factors play important roles in the control of cell elongation. Two bHLH proteins, PACLOBTRAZOL RESISTANCE1 (PRE1) and Arabidopsis ILI1 binding bHLH1 (IBH1), antagonistically regulate cell elongation in response to brassinosteroid and gibberellin signaling, but the detailed molecular mechanisms by which these factors regulate cell elongation remain unclear. Here, we identify the bHLH transcriptional activators for cell elongation (ACEs) and demonstrate that PRE1, IBH1, and the ACEs constitute a triantagonistic bHLH system that competitively regulates cell elongation. In this system, the ACE bHLH transcription factors directly activate the expression of enzyme genes for cell elongation by interacting with their promoter regions. IBH1 negatively regulates cell elongation by interacting with the ACEs and thus interfering with their DNA binding. PRE1 interacts with IBH1 and counteracts the ability of IBH1 to affect ACEs. Therefore, PRE1 restores the transcriptional activity of ACEs, resulting in induction of cell elongation. The balance of triantagonistic bHLH proteins, ACEs, IBH1, and PRE1, might be important for determination of the size of plant cells. The expression of IBH1 and PRE1 is regulated by brassinosteroid, gibberellins, and developmental phase dependent factors, indicating that two phytohormones and phase-dependent signals are integrated by this triantagonistic bHLH system.

Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance.

Many eukaryotes have from one to three heat shock factors (Hsfs), but plants have more than 20 Hsfs, designated class A, B, and C. Class A Hsfs are activators of transcription, but details of the roles of individual Hsfs have not been fully characterized. We show here that Arabidopsis (Arabidopsis thaliana) HsfB1 and HsfB2b, members of class B, are transcriptional repressors and negatively regulate the expression of heat-inducible Hsfs (HsfA2, HsfA7a, HsfB1, and HsfB2b) and several heat shock protein genes. In hsfb1 hsfb2b double mutant plants, the expression of a large number of heat-inducible genes was enhanced in the non-heat condition (23°C) and the plants exhibited slightly higher heat tolerance at 42°C than the wild type, similar to Pro35S:HsfA2 plants. In addition, under extended heat stress conditions, expression of the heat-inducible Hsf genes remained consistently higher in hsfb1 hsfb2b than in the wild type. These data indicate that HsfB1 and HsfB2b suppress the general heat shock response under non-heat-stress conditions and in the attenuating period. On the other hand, HsfB1 and HsfB2b appear to be necessary for the expression of heat stress-inducible heat shock protein genes under heat stress conditions, which is necessary for acquired thermotolerance. We show that the heat stress response is finely regulated by activation and repression activities of Hsfs in Arabidopsis.

Arabidopsis WUSCHEL is a bifunctional transcription factor that acts as a repressor in stem cell regulation and as an activator in floral patterning.

Most transcription factors act either as activators or repressors, and no such factors with dual function have been unequivocally identified and characterized in plants. We demonstrate here that the Arabidopsis thaliana protein WUSCHEL (WUS), which regulates the maintenance of stem cell populations in shoot meristems, is a bifunctional transcription factor that acts mainly as a repressor but becomes an activator when involved in the regulation of the AGAMOUS (AG) gene. We show that the WUS box, which is conserved among WOX genes, is the domain that is essential for all the activities of WUS, namely, for regulation of stem cell identity and size of floral meristem. All the known activities of WUS were eliminated by mutation of the WUS box, including the ability of WUS to induce the expression of AG. The mutation of the WUS box was complemented by fusion of an exogenous repression domain, with resultant induction of somatic embryogenesis in roots and expansion of floral meristems as observed upon ectopic expression of WUS. By contrast, fusion of an exogenous activation domain did not result in expanded floral meristems but induced flowers similar to those induced by the ectopic expression of AG. Our results demonstrate that WUS acts mainly as a repressor and that its function changes from that of a repressor to that of an activator in the case of regulation of the expression of AG.

Arabidopsis zinc finger homeodomain transcription factor BRASSINOSTEROID-RELATED HOMEOBOX 2 acts as a positive regulator of brassinosteroid response.

The brassinosteroid (BR) phytohormone is an important regulator of plant growth. To identify novel transcription factors that regulate BR responses, we screened chimeric repressor gene silencing technology (CRES-T) plants, in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, with brassinazole (Brz), an inhibitor of BR biosynthesis. We identified that a line that expressed the chimeric repressor for zinc finger homeobox transcription factor, BRASSINOSTEORID-RELATED-HOMEOBOX-2 (BHB2-sx), exhibited Brz-hypersensitive phenotype with shorter hypocotyl under dark, dwarf and round and dark green leaves similar to BR-deficient phenotype. Similar to BHB2-sx plants, bhb2 knockout mutant also exhibited Brz hypersensitive phenotype. In contrast, ectopic expression of BHB2 (BHB2-ox) showed hypocotyl elongation phenotype (BR excessive), showing decrease to Brz sensitivity. The expression of the DWF4 and CPD BR biosynthesis genes was repressed in BHB2-sx plants, whereas it was enhanced in BHB2-ox plants. The BR deficient-like phenotype of BHB2-sx plants was partially restored by treatment with brassinolide (BL), indicating that the BR deficient phenotype of BHB2-sx plant may be due to suppression of BR biosynthesis. Our results indicate that BHB2 is a positive regulator of BR response may be due to the promotion of BR biosynthesis genes.

Arabidopsis homeobox-leucine zipper transcription factor BRASSINOSTEROID-RELATED HOMEOBOX 3 regulates leaf greenness by suppressing BR signaling.

Brassinosteroid (BR) is a phytohormone that acts as important regulator of plant growth. To identify novel transcription factors that may be involved in unknown mechanisms of BR signaling, we screened the chimeric repressor expressing plants (CRES-T), in which transcription factors were converted into chimeric repressors by the fusion of SRDX plant-specific repression domain, to identify those that affect the expression of BR inducible genes. Here, we identified a homeobox-leucine zipper type transcription factor, BRASSINOSTEROID-RELATED-HOMEOBOX 3 (BHB3), of which a chimeric repressor expressing plants (BHB3-sx) significantly downregulated the expression of BAS1 and SAUR-AC1 that are BR inducible genes. Interestingly, ectopic expression of BHB3 (BHB3-ox) also repressed the BR inducible genes and shorten hypocotyl that would be similar to a BR-deficient phenotype. Interestingly, both BHB3-sx and BHB3-ox showed pale green phenotype, in which the expression of genes related photosynthesis and chlorophyll contents were significantly decreased. We found that BHB3 contains three motifs similar to the conserved EAR-repression domain, suggesting that BHB3 may act as a transcriptional repressor. These results indicate that BHB3 might play an important role not only to the BR signaling but also the regulation of greenings.

HsfA1d and HsfA1e involved in the transcriptional regulation of HsfA2 function as key regulators for the Hsf signaling network in response to environmental stress.

Heat shock transcription factor A2 (HsfA2) acts as a key component of the Hsf signaling network involved in cellular responses to various types of environmental stress. However, the mechanism governing the regulation of HsfA2 expression is still largely unknown. We demonstrated here that a heat shock element (HSE) cluster in the 5'-flanking region of the HsfA2 gene is involved in high light (HL)-inducible HsfA2 expression. Accordingly, to identify the Hsf regulating the expression of HsfA2, we analyzed the effect of loss-of-function mutations of class A Hsfs on the expression of HsfA2 in response to HL stress. Overexpression of an HsfA1d or HsfA1e chimeric repressor and double knockout of HsfA1d and HsfA1e Arabidopsis mutants (KO-HsfA1d/A1e) significantly suppressed the induction of HsfA2 expression in response to HL and heat shock (HS) stress. Transient reporter assays showed that HsfA1d and HsfA1e activate HsfA2 transcription through the HSEs in the 5'-flanking region of HsfA2. In the KO-HsfA1d/A1e mutants, 560 genes, including a number of stress-related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1 and HsfB2a, were down-regulated compared with those in the wild-type plants under HL stress. The PSII activity of KO-HsfA1d/A1e mutants decreased under HL stress, while the activity of wild-type plants remained high. Furthermore, double knockout of HsfA1d and HsfA1e impaired tolerance to HS stress. These findings indicated that HsfA1d and HsfA1e not only regulate HsfA2 expression but also function as key regulators of the Hsf signaling network in response to environmental stress.

Surgical repair of hepatic hydrothorax caused by diaphragmatic fistula.

A 65-year-old woman visited our hospital complaining of dyspnea several days before admission. A chest X-ray showed massive right-sided pleural effusion, which was not observed 1 month previously. Although the patient had never been diagnosed with cirrhosis at regular visits, the patient was diagnosed with primary biliary cholangitis at admission. Hepatic hydrothorax was suspected because pleural effusion was transudative. A diaphragmatic fistula was confirmed and closed by thoracoscopy. Pleural effusion did not reappear after this procedure. Existence of a diaphragmatic defect should be confirmed under direct vision if pleural effusion accumulates acutely or becomes beyond control.

Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana.

Yeast one-hybrid screening is widely used for the identification of transcription factors (TFs) that interact with specific DNA sequences. However, screening a whole cDNA library is not efficient for the identification of TFs because TF genes represent only a small percentage of clones in a cDNA library. Here, we present the development of an efficient yeast one-hybrid screening system using a prey library composed only of approximately 1,500 TF cDNAs of Arabidopsis thaliana. This library enabled us to isolate a TF that binds to a specific promoter sequence with high efficiency, even when the promoter region of the gene of interest was directly employed as bait. Furthermore, this library was also successfully applied as a yeast two-hybrid library to find TFs that interact with specific proteins. This efficient system will contribute to the elucidation of gene regulatory networks in plants.

Analysis of BAC clones containing homologous sequences on the end of the Xq arm and on chromosome 7 in the dioecious plant Silene latifolia.

Silene latifolia is a model dioecious plant with morphologically distinguishable XY sex chromosomes. The end of the Xq arm is quite different from that of the Yp arm, although both are located at opposite ends of their respective chromosomes relative to a pseudo-autosomal region. The Xq arm does not seem to originate from the same autosome as the Yp arm. Bacterial artificial chromosome clone #15B12 has an insert containing a 130-kb stretch in which a 313-bp satellite DNA is repeated 420 times. PCR with a single primer revealed that this 130-kb stretch consists of three reversals of the orientation of the satellite DNA. A non-long terminal repeat retroelement and two sequences that share homology with an Oryza sativa RING zinc finger and a putative Arabidopsis thaliana protein, respectively, were found in the sequences that flank the satellite DNA. Fluorescence in situ hybridization carried out using this low-copy region of #15B12 as a probe confirmed that these sequences originated from the X chromosome and that homologous sequences exist at the end of chromosome 7. The region distal to DD44X on the Xq arm is postulated to have recombined with a region containing satellite DNA on chromosome 7 during the process of sex chromosome evolution.

Improving the Efficiency of Adventitious Shoot Induction and Somatic Embryogenesis via Modification of WUSCHEL and LEAFY COTYLEDON 1.

The induction of adventitious organs, such as calli, shoots, and somatic embryos, in tissue culture is a useful technique for plant propagation and genetic modification. In recent years, several genes have been reported to be adventitious organ inducers and proposed to be useful for industrial applications. Even though the Arabidopsis (Arabidopsis thaliana) WUSCHEL (WUS) and LEAFY COTYLEDON 1 (LEC1) genes can induce adventitious organ formation in Arabidopsis without phytohormone treatment, further improvement is desired. Here, we show that modifying the transcriptional repression/activation activities of WUS and LEC1 improves the efficiency of adventitious organ formation in Arabidopsis. Because WUS functions as a transcriptional repressor during the induction of adventitious organs, we fused it to an artificial strong repression domain, SUPERMAN REPRESSION DOMAIN X (SRDX). Conversely, we fused the strong transcriptional activation domain VP16 from herpes simplex virus to LEC1. Upon overexpression of the corresponding transgenes, we succeeded in improving the efficiency of adventitious organ induction. Our results show that the modification of transcriptional repression/activation activity offers an effective method to improve the efficiency of adventitious organ formation in plants.

A novel group of transcriptional repressors in Arabidopsis.

We showed previously that the ERF-associated amphiphilic repression (EAR) motif is a plant-specific repression domain that contains the conserved amino acid sequence LXLXL. In this report, we describe the identification of a novel repression domain, L/VR/KLFGVXM/V/L, which is different from known EAR motifs, in B3 DNA-binding domain transcription factors in Arabidopsis. Database analysis revealed that 29 Arabidopsis transcription factors, which included members of the RAV, ARF, Hsf and MYB families, contain the R/KLFGV conserved motif found in the novel repression domain. We demonstrated that factors that contain the R/KLFGV motif, namely, RAV1, RAV2, HsfB1 and HsfB2b, exhibited the repressive activity.

Primary pleural synovial sarcoma with repeated resection leading to long-term survival.

Primary pleural synovial sarcoma is a malignant tumour and thought to be more aggressive than synovial sarcoma which occurs in extremities. Its treatment strategy has not been fully established because of its rarity. We report a primary pleural synovial sarcoma case which achieved a long-term survival with repeated surgery of recurrent pleural tumour. A 39-year-old man presented with a gradually enlarged tumour in the left hemithorax. The tumour was resected and diagnosed as primary pleural synovial sarcoma. The tumour was slowly growing and repeatedly recurrent in the left pleura. The surgical resections for the recurrent tumours were performed 6 years and 11 years after the initial surgery. Intriguingly, recurrent tumour which developed after second surgery exhibited temporally spontaneous regression. Our patient remains alive 12 years after the initial surgery. Repeated resection of metastatic lesion can achieve long survival in primary pleural synovial sarcoma.

Sugar-responsive transcription factor bZIP3 affects leaf shape in Arabidopsis plants.

Sugars are essential for plant metabolism, growth and development. Plants must therefore manage their growth and developmental processes in response to sugar availability. Sugar signaling pathways constitute a complicated molecular network and are associated with global transcriptional regulation. However, the molecular mechanisms underlying sugar signaling remain largely unclear. This study reports that the protein basic-region leucine zipper 3 (bZIP3) is a novel sugar-responsive transcription factor in Arabidopsis plants. The expression of bZIP3 was rapidly repressed by sugar. Genetic analysis indicated that bZIP3 expression was modulated by the SNF1-RELATED KINASE 1 (SnRK1) pathway. Moreover, transgenic plants overexpressing bZIP3 and dominant repressor form bZIP3-SRDX showed aberrant shaped cotyledons with hyponastic bending. These findings suggest that bZIP3 plays a role in plant responses to sugars and is also associated with leaf development.