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Original Research ARTICLE
The polyamine spermine induces the unfolded protein response via the MAPK cascade in Arabidopsis
- 1Graduate School of Life Sciences, Tohoku University, Sendai, Japan
- 2Biodiversity and Climate Research Center, Laboratory Center, Frankfurt am Main, Germany
- 3Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Japan
- 4Faculty of Pharmaceutical Sciences, Josai University, Sakado, Japan
In Arabidopsis three basic region leucine zipper (bZIP) transcription factor genes, bZIP17, bZIP28, and bZIP60, play crucial roles in the unfolded protein response (UPR). Previously we found that bZIP60 is one of the spermine-induced genes. Consequently we further investigated the response of all the three bZIP genes to spermine. Expression of bZIP17, bZIP28, and bZIP60, and also their target genes was activated by spermine application as well as in plants with elevated endogenous spermine levels. Furthermore, spermine activated the splicing of the bZIP60 transcript mediated by the ribonuclease activity of inositol-requiring enzyme 1 and also recruited bZIP17 and bZIP60 proteins from endoplasmic reticulum to nucleus. We therefore propose that spermine is a novel UPR inducer. Moreover, induction of UPR by spermine required calcium-influx to the cytoplasm and the genes for mitogen-activated protein kinase kinase 9 (MKK9), mitogen-activated protein kinase 3 (MPK3) and MPK6. The result indicates that spermine-induced UPR is mediated by the MKK9-MPK3/MPK6 cascade in Arabidopsis.
Polyamines (PAs) are low-molecular mass, aliphatic compounds and they are ubiquitously present in all living organisms (Tabor and Tabor, 1984; Cohen, 1998). In plants, major PAs are putrescine (Put), spermidine (Spd), spermine (Spm), and thermospermine (T-Spm; Knott et al., 2007; Takano et al., 2012). PAs have important roles in various physiological processes not only in embryogenesis, growth, development and aging/senescence but also in adaptation responses to abiotic and biotic stresses (Kusano et al., 2008; Alcázar et al., 2010; Takahashi and Kakehi, 2010; Tiburcio et al., 2014; Berberich et al., 2015).
Polyamine precursors are ornithine and/or arginine. Ornithine is converted to Put by ornithine decarboxylase (ODC), while arginine is converted to Put via agmatine by three steps of enzyme reactions; arginine decarboxylase (ADC), agmatine iminohydrolase, and N-carbamoylputrescine amidohydrolase. Put is converted to Spd by Spd synthase, in which another substrate, decarboxylated S-adenosylmethionine (dcSAM), is required. dcSAM is synthesized by S-adenosylmethionine decarboxylase (SAMDC) from S-adenosylmethionine (SAM). Spd is further converted either to Spm by Spm synthase (SPMS) or to T-Spm by T-Spm synthase, also called ACAULIS5 (ACL5). SPMS and ACL5 also require dcSAM as another substrate (Kusano et al., 2008; Alcázar et al., 2010; Takahashi and Kakehi, 2010). In Arabidopsis, the ODC gene is absent (Hanfrey et al., 2001). Thus Put is synthesized only by the ADC pathway in this organism. On the other hand, PA catabolism is governed by two enzymes: the one is copper-dependent amine oxidase (CuAO) and the other is polyamine oxidase (PAO; Kusano et al., 2015). In Arabidopsis, there are more than 10 CuAOs and 5 PAOs. All the latter enzymes are characterized (Kusano et al., 2015).
As mentioned, involvement of PAs and their metabolism in the defense responses against pathogens is described (Walters, 2003a,b). PAs and their oxidative products by CuAO and PAO play crucial roles in plant defense responses (Berberich et al., 2015, and the references therein). When Nicotiana tabacum carrying the resistance gene N was infected by Tobacco mosaic virus (TMV), host plant displayed a hypersensitive response (HR). Once HR occurs, it is known that the tissues surrounding the TMV infection sites were killed by a suicidal reaction to prevent the virus multiplication. Yamakawa et al. (1998) reported that Spm enriched in the apoplastic space during HR triggered by N. tabacum-TMV pathosystem. Subsequently we found that exogenously applied Spm stimulates the phosphorylation activities of two MAPKs, wound-induced protein kinase (WIPK, Seo et al., 1995) and salicylic acid-induced protein kinase (SIPK, Zhang and Klessig, 1997), which trigger the expression of downstream defense genes in N. tabacum (Takahashi et al., 2003, 2004), and proposed it to call 'Spm-signaling pathway.' Massive identification of the Spm-responsive genes using a super serial analysis of gene expression (SuperSAGE) approach was performed in Arabidopsis thaliana (Mitsuya et al., 2009). The identified Spm-responsive genes behaved similarly during cucumber mosaic virus (CMV)-induced HR in A. thaliana (Takahashi et al., 1994; Mitsuya et al., 2009). One of them was a gene (bZIP60) encoding a basic region leucine zipper 60 protein. bZIP60 was reported as a key transcription factor (TF) gene which is involved in the unfolded protein response (UPR; Iwata and Koizumi, 2005). UPR is induced, when unfolded or misfolded proteins are accumulated in the endoplasmic reticulum (ER), to refold or degrade the corresponding proteins (Walter and Ron, 2011). In plants, three bZIP-type proteins, bZIP17, bZIP28 as well as bZIP60, govern UPR in Arabidopsis (Iwata et al., 2008; Liu and Howell, 2010; Iwata and Koizumi, 2012; Howell, 2013). Those bZIP proteins reside in the ER under unstressed conditions, and once they sense ER stress, the former two proteins move to the Golgi apparatus and migrate to the nucleus after proteolytic processing by site 1 and site 2 proteases (S1P and S2P; Liu et al., 2007a,b). In contrast, bZIP60 transcript is unconventionally spliced by an RNase activity of inositol-requiring enzyme 1 (IRE1) and the product of the resulting spliced bZIP60 transcript is recruited to the nucleus. The Arabidopsis genome contains two IRE1 genes, AtIRE1A (At2g17520) and AtIRE1B (At5g24360; Liu and Howell, 2010; Deng et al., 2011; Nagashima et al., 2011; Iwata and Koizumi, 2012; Srivastava et al., 2013). Several chemicals such as tunicamycin (TM) and dithiothreitol (DTT) are known to be UPR inducers. TM inhibits the N-linked glycosylation and DTT inhibits the disulfide bond formation, both of which are important for protein maturation.
Here, we aim to reveal whether Spm only acts on bZIP60 or induces the whole UPR in Arabidopsis. We also address the upstream components in Spm-induced UPR pathways.
Materials and Methods
Plant Materials and Growth Condition
Arabidopsis thaliana ecotype Columbia-0 (Col-0) is used as wild type (WT). T-DNA insertion mutants, ire1a (SALK_018112, Deng et al., 2011, 2013), ire1b (SAIL_238_F07, Deng et al., 2011, 2013), bzip60 (SALK_050203, Deng et al., 2011, 2013), Atmpk6 (SALK_127507, Yoo et al., 2008; Zhou et al., 2009), Atmpk3 (SALK_151594, Yoo et al., 2008; Zhou et al., 2009) were obtained from Dr. Stephen H. Howell (Iowa State University) and Arabidopsis Biological Resource Center, while Atmkk9 (SAIL_60_H06, Xu et al., 2008) was kindly provided from Dr. Dongtao Ren (China Agricultural University). The double mutant, ire1aire1b, was obtained by crossing of ire1a and ire1b. Seeds were surface sterilized by 70% ethanol for 1min, then by 1% sodium hypochlorite solution containing 0.1% Tween-20 for 15 min, then followed by rinsing with sterilized water, three times. Sterilized seeds were sown on half strength MS medium (1% w/v agar, 1% w/v sucrose, 0.5 x MS salts, 0.05% vitamin B5, pH 5.6) and incubated in a growth chamber at 22°C under 16 h light/8 h dark conditions.
Various Treatments
Ten-days-old Arabidopsis seedlings were incubated in half strength MS solution supplemented with or without Spm (0.5 mM) for 12 h under continuous light condition. PAs treatment: 0.5 mM concentration of PAs was used unless otherwise mentioned and incubated for 12 h. DTT treatment: DTT was applied at 2 mM concentration for 5 h in half strength MS solution. Lanthanum chloride (LaCl3) treatment: 10-days-old seedlings were incubated with or without La3+ (0.5 mM) for 12 h.
Genome DNA-Polymerase Chain Reaction (PCR) and Reverse Transcription (RT)-PCR Analyses
Plant genome DNA was prepared by the procedure described by Murray and Thompson (1980). Total RNA was extracted from the respective plants samples using Sepasol-RNA I Super (Nacalai Tesque, Kyoto, Japan). Total RNA was treated with DNaseI (Takara DNase, Japan). First strand cDNA was synthesized from the DNaseI-treated RNA with ReverTra Ace (Toyobo Co. Ltd., Osaka, Japan), oligo-dT primer and dNTPs. AtActin was amplified using a specific primer pair (Supplementary Table S1) and used as a loading control. The amplified DNA fragments were separated by agarose-gels and visualized by ethidium bromide-staining.
Quantitative Real-Time RT-PCR
Quantitative RT-PCR (qRT-PCR) analysis was performed by StepOne Real-Time PCR System (Applied Biosystems) using SYBR® Green RT-PCR Kit (FastStart Universal SYBR Green Master, ROX). A standard curve was constructed from different genes and the values were normalized to Actin levels. The primers used for qRT-PCR were described in Supplementary Table S1.
bZIP60 Splicing Assay
bZIP60 splicing assay was performed using the following primers; for detecting a unspliced form (SPU) and a spliced form (SPS), bZIP60-F primer and bZIP60-UB1 reverse primer and bZIP60-F primer and bZIP60-SB2 reverse primer, respectively, were used (Supplementary Table S1).
Construction of Green Fluorescent Protein (GFP) Fusion Plasmids, Biolistic Bombardments, and Microscopic Observation
The basal GFP vector was constructed as described previously (Ono et al., 2012). Briefly, a GFP coding fragment was amplified by PCR using pGFP2 (provided by Dr. N.-H. Chua). The fragment was double-digested with BamHI and SmaI, and subcloned into the pBI221 vector (Invitrogen), yielding pBI221GFP. Next, the internal two SacI sites of AtbZIP17 coding region were mutated without changing the amino acid sequence by two-step PCR. Then, a second PCR was performed on the respective mixtures of the first PCR products using the primers, AtbZIP17-SmaI-F and AtbZIP17-SacI-Rv (Supplementary Table S1). The coding region of AtbZIP60 was amplified by PCR. The resulting AtbZIP17 and AtbZIP60 fragments digested with SmaI and SacI were subcloned into the corresponding restriction enzyme sites of pBI221GFP, yielding pBI221GFP-AtbZIP17 and pBI221GFP-AtbZIP60, respectively. The resulting GFP-AtbZIPs' constructs were bombarded into onion bulbs by particle bombardment. ER-targeting DsRED plasmid (Okamoto et al., 2008) or nuclear-targeting mCherry-VirD2_NLS plasmid (CD3-1106, purchased from ABRC) was co-bombarded as the ER- and nuclear-markers. After incubating the onion bulbs with or without Spm (or DTT) at 22°C for 16 h under darkness, onion epidermal cells were peeled and placed onto glass slides and observed with a fluorescence microscope (BX61; Olympus).
Arabidopsis Transgenic Plants Overexpressing SPMS
Three independent lines of the transgenic Arabidopsis overexpressing SPMS were used. The detailed procedure to generate the transgenics was described in Sagor et al. (2012a).
PA Analysis
Polyamine analysis was performed as described in Naka et al. (2010). In brief, plant samples (0.3–0.5 g per sample) were pulverized with a mortar and pestle under liquid nitrogen. Five volumes (2.5 mL per 0.5 g of plant sample) of 5% (v/v) cold perchloric acid were added to the resulting fine powders. The mixtures were transferred to plastic tubes and kept on ice for 1 h. After centrifugation at 15,000 × g for 30 min at 4°C, the supernatants were combined and filtered using a filter syringe (pore size, 0.2 μm). One milliliter of 2 N NaOH was added to 1.5 mL of plant extract and mixed thoroughly. Then 10 μL of benzoyl chloride was added and the mixture was incubated at room temperature for 20 min. After adding 2 mL of saturated NaCl and 2 mL of diethyl ester, samples were vigorously mixed and then centrifuged at 3,000 × g for 10 min at 4°C for phase-separation. An aliquot (1.5 mL) of the organic solvent phase was evaporated and the residue was resuspended in 50 μL of methanol. Benzoylated PAs were analyzed with a programmable Agilent 1200 liquid chromatograph using a reverse-phase column (4.6 mm × 250 mm, TSK-GEL ODS-80Ts, TOSOH, Tokyo, Japan) and detected at 254 nm. One cycle of the run took 60 min in total with a flow rate of 1 mL/min at 30°C, i.e., 42% acetonitrile for 25 min for PA separation, increased to 100% acetonitrile over 3 min, 100% acetonitrile for 20 min for washing, decreased to 42% acetonitrile over 3 min, and finally 42% acetonitrile for 9 min.
Statistical Analysis
Student's t-tests were used for statistical analysis and were performed using Microsoft Excel statistical tools.
Exogenously Applied Spm Induces bZIP17 and bZIP28 as Well as bZIP60 and their Target Genes
Ten-days-old Arabidopsis ecotype (Col-0) seedlings were treated with Spm or DTT (as positive treatment for UPR induction) and qRT-PCR was performed. Spm induced bZIP17, bZIP28 and bZIP60, and their respective target genes, HB-7, CNX1, and BiP3, respectively, as similar as DTT did (Figure 1, Supplementary Figure S1). The result clearly indicates that Spm is a novel UPR inducer.
FIGURE 1. Exogenous Spm induces the expression of bZIP17, bZIP28, and bZIP60 and their target genes. qRT-PCR was performed using the cDNAs prepared from control- and Spm-treated Arabidopsis seedlings. (A) bZIP17; (B) HB-7; (C) bZIP28; (D) CNX1; (E) bZIP60; (F) Bip3. The transcript level in control Arabidopsis plant was set as 1 and relatively displayed. Values are mean (+SD) from three independent experiments. Asterisks indicate significant difference (∗∗P < 0.01).
Spm Activates the IRE1-Mediated bZIP60 Splicing
Canonical UPR inducers activate bZIP60 splicing, which is mediated by IRE1 (Deng et al., 2011, 2013). Thus we addressed this point. At 1 h after Spm treatment, the unspliced form (SPU) of bZIP60 transcripts was accumulated. At a little time gap after the SPU detection, the spliced form (SPS) of bZIP60 transcripts was detected, which followed by BiP3 induction (Figure 2). To further prove the IRE1 involvement in the process, we used ire1a, ire1b and ire1a ire1b (see Supplementary Figure S2) double mutant along with bzip60 mutant (Deng et al., 2011, 2013). In ire1a mutant, the levels of the SPS form of bZIP60 transcript and BiP3 transcript were slightly lowered compared to those of WT after Spm treatment. In ire1b and ire1a ire1b mutants, the transcriptional attenuation of the SPS-bZIP60 and BiP3 was strikingly observed (Figure 3). It shows that IRE1B is mainly participated in Spm-induced bZIP60 unconventional splicing.
FIGURE 2. Time-course analysis of bZIP60 splicing upon Spm treatment. (A) A schematic representation of bZIP60 transcript with the position and orientation of primer pairs used for detecting the unspliced (SPU)- and spliced (SPS)-forms of bZIP60. (B) Time course RT-PCR analysis of SPU and SPS forms of bZIP60 transcript and its downstream target BiP3 transcript at indicated time intervals in control and Spm-treated seedlings. Actin was used as an internal control.
FIGURE 3. Spm-induced bZIP60 splicing occurs in an IRE1-dependent manner. Ten-days-old seedlings of Col-0 (control), ire1a (SALK_018112, Deng et al., 2011, 2013; Nagashima et al., 2011) mutant, ire1b (SAIL_238_F07) mutant, and ire1aire1b double mutant, and bzip60 (SALK_050203) mutant were treated with half strength MS solution as a control (-) and half strength MS solution containing 0.5 mM Spm (+). Results of RT-PCR for expression analysis of SPU and SPS forms of bZIP60 and its downstream gene BiP3 were displayed. Actin was used as an internal control.
Endogenous Spm Induces bZIP17, bZIP28, and bZIP60 and their Target Genes
We have generated the transgenic Arabidopsis overexpressing SPMS (Sagor et al., 2012a). The three independent lines, designated as SPMS OX_1, SPMS OX_15, and SPMS OX_21, were used in this study. In those transgenics, the SPMS transcripts were remarkably accumulated (Supplementary Figure S3A) and concomitantly Spm content increased significantly, in contrast, Spd content decreased (Supplementary Figure S3B). We examined the transcript levels of bZIP17, bZIP28, and bZIP60 in the Spm-enriched Arabidopsis plants. The levels of bZIP17, bZIP28, and bZIP60 transcripts became approximately 1.2∼1.3-fold, 2- to 3-fold, and 9- to 12-fold, respectively, in the SPMS OX transgenics compared those in WT (Figures 4A,C,E). The levels of BiP3 transcripts became clearly higher than those in WT (Figure 4F), whereas those of HB-7 and CNX1 were still higher than those in WT but not so much significant (Figures 4B,D). In the Spm-enriched plants, homeostatic regulation to suppress the constitutive UPR may be operated. Anyway, not only exogenously applied Spm but also high endogenous Spm are able to induce the UPR. PA specificity assay showed that T-Spm has a similar inducing activity as Spm does, and Spd has a weaker activity but Put has no such activity (Sagor et al., 2012b; Supplementary Figure S4, data not shown). Spm and T-Spm induced the expression of bZIP60 even at 100 μM (Supplementary Figure S4).
FIGURE 4. Endogenous Spm induces the expression of bZIP17, bZIP28, and bZIP60 and their target genes. The levels of bZIP17 (A), bZIP28 (C), and bZIP60 (E) transcripts, and their respective target gene transcripts [HB-7 (B), CNX1 (D), and BiP3 (F)] in the SPMS_OX transgenic lines were quantitatively determined by qRT-PCR analysis using the primer pairs listed in Supplementary Table S1. Values are mean (+SD) from three independent experiments. Asterisks indicate significant difference (∗P < 0.05 and ∗∗P < 0.01).
Spm Recruits bZIP17 and bZIP60 from ER to Nucleus
bZIP17, bZIP28, and bZIP60 encode the bZIP-type TF proteins. To accomplish their roles as TFs, their products have to reach to nuclei. Here we generated the GFP:bZIP17 and GFP:bZIP60 fusion plasmids and delivered them to onion epidermal cells. Before Spm treatment, both the fusion proteins seemed to locate in ER because the green fluorescent signals overlapped with the red fluorescent signals emitted by the ER-positive marker (Figures 5Aa–c,Ba–c). Upon Spm treatment, the green fluorescent signals from both the fusion proteins were merged to the red fluorescent signals emitted from the nucleus-targeting marker plasmid, indicating that bZIP17 and bZIP60 proteins were recruited to nucleus after Spm treatment (Figures 5Ag–i, Bg–i) as well as after DTT treatment (Figures 5Ad–f,Bd–f).
FIGURE 5. Recruitment of bZIP17 and bZIP60 proteins from ER to nucleus upon Spm treatment. (A) GFP:bZIP17 fusion protein under normal condition (a–c); DTT treatment (d–f); and Spm treatment (g–i). (B) GFP:bZIP60 fusion protein under normal condition (a–c); DTT treatment (d–f); and Spm treatment (g–i). The plasmids encoding GFP:bZIP17 and GFP:bZIP60 proteins were co-bombarded to onion bulbs with the plasmid encoding an ER marker peptide tagged with Ds-Red or with the plasmid encoding a nuclear marker protein VirD2_NLS using a particle gun. The bombarded samples were treated with or without Spm for 16 h under darkness at 22°C. The samples were also treated with DTT as a positive reference. Then onion epidermal cell layers were peeled off and observed by a fluorescence microscope (BX61; Olympus). Bar indicates 10 μm.
Ca2+-Influx is One of the Upstream Components of Spm-Induced UPR
In tobacco plant, we have demonstrated that Ca2+-influx to cytoplasm and the activation of the mitogen-activated protein kinase (MAPK) cascade consisting of NtMEK2-SIPK/WIPK are prerequisite for triggering a Spm-signaling pathway (Takahashi et al., 2003, 2004). First, therefore, we addressed whether Ca2+-influx to cytoplasm is required for UPR induction by Spm treatment. We applied a Ca2+-channel blocker, La3+, to the plant samples when Spm was applied. Upregulation of bZIP17, bZIP28, and bZIP60 was totally alleviated in the plants co-treated with Spm and La3+ (Figures 6A,C,E). Induction of the target genes, HB-7, CNX1 and BiP3, of bZIP17, bZIP28, and bZIP60 was also blocked by La3+ treatment (Figures 6B,D,F). The results indicate that Spm-induced UPR pathway requires the enhanced Ca2+ influx to cytoplasm.
FIGURE 6. Ca2+-channel blocker alleviates the UPR induction by Spm. qRT-PCR was performed using the cDNAs prepared from control-, Spm-treated, and Spm and La3+-co-treated Arabidopsis seedlings. (A) bZIP17; (B) HB-7; (C) bZIP28; (D) CNX1; (E) bZIP60; (F) Bip3. The transcript level in control Arabidopsis plant was set as 1 and relatively displayed. Values are mean (+SD) from three independent experiments. Asterisks indicate significant difference (∗∗P < 0.01).
A MAPK Cascade is Another Upstream Component of Spm-Induced UPR
In A. thaliana, there are more than 60 MAPK kinase kinases (MAP3K), 10 MAPKKs (MKK), and 20 MAPKs (MAPK Group, 2002). Therefore, we examined whether any MKKs are up-regulated by exogenously applied Spm. Of 10 MKKs, MKK9 was strikingly induced by Spm (Figure 7A). The levels of MKK4 and MKK5 transcripts were also accumulated at the lesser extent compared to those of MKK9 (Figure 7A). The previous research showed that the downstream MAPKs of MKK9 are MPK6 (SIPK ortholog) and/or MPK3 (WIPK ortholog; see review Colcombet and Hirt, 2008; Yoo et al., 2008; Zhou et al., 2009; Rodriguez et al., 2010). In addition to MKK9, MPK3 was identified as Spm-responsive gene in a SuperSAGE analysis (Mitsuya et al., 2009). So we tested the expressional response of MPK3 and MPK6 upon Spm treatment. The qRT-PCR result showed that expression of both MPK3 and MPK6 was up-regulated by Spm treatment (Figure 7B). Furthermore, we quantified the transcript levels of MKK9, MPK3, and MPK6 in the Spm-enriched transgenic plants, and found that MKK9 and MPK3 transcripts were distinctly accumulated and MPK6 transcripts increased to approximately 1.2–1.4-fold levels in the SPMS OX plants compared to those in WT (Figure 7C).
FIGURE 7. Spm upregulates MKK9, MPK3, and MPK6. (A) Exogenous Spm upregulated MKK9 among 10 MKKs. (B) Exogenous Spm upregulated MPK3 and MPK6. (C) The transcript levels of MKK9, MPK3, and MPK6 in the SPMS_OX transgenic lines. The levels of MKK9, MPK3, and MPK6 transcripts were quantitatively determined by qRT-PCR using the primer pairs listed in Supplementary Table S1. Values are mean (+SD) from three independent experiments. Asterisks indicate significant difference (∗P < 0.05 and ∗∗P < 0.01).
Spm-Induction of UPR Pathway is Mediated by the MKK9-MPK3/MPK6 Cascade
Then the question arose whether all the paths of bZIP17, bZIP28, and bZIP60 in the UPR are positioned downstream of the MKK9-MPK3/MPK6 cascade. To answer the issue, we used the T-DNA insertion mutants of MKK9, MPK3, and MPK6. The T-DNA insertion sites in MKK9, MPK3, and MPK6 were displayed in Supplementary Figure S5A. The homogeneity of the T-DNA insertion in the mutants, mkk9, mpk3, and mpk6, was confirmed by the genome DNA-PCR (Supplementary Figures 5B–D; Xu et al., 2008; Yoo et al., 2008; Zhou et al., 2009). The levels of MKK9-, MPK3-, and MPK6-transcripts in the mkk9, mpk3, and mpk6 mutants were almost null in relative to those of WT, respectively (Supplementary Figures 5E–G). Using the loss-of-function mutants, we examined the levels of bZIP17, bZIP28, and bZIP60 transcripts upon Spm treatment. The expression of bZIP17, bZIP28, and bZIP60 was not enhanced by Spm treatment in the mkk9 mutant (Figure 8A). The expression of their target genes was also not induced by Spm in the mkk9 mutant (Figure 8B). The expression of the bZIP genes and their target genes was more or less similarly induced by Spm in the mpk3 and mpk6 mutants. The latter result may be explained that MPK3 and MPK6 have redundant function to transduce the Spm-signal. It should be noted that Spm-signaling pathway was blocked in Nicotiana benthamiana in which both, SIPK and WIPK, were simultaneously silenced but neither in SIPK-silencing nor in WIPK-silencing N. benthamiana (Takahashi et al., 2004). Collectively it indicates that the induction of bZIP17, bZIP28, and bZIP60 depends on the MKK9-MPK3/MPK6 cascade.
FIGURE 8. The expression of bZIP17, bZIP28, and bZIP60 (A) and their target genes (B) in the loss-of-function mutants, mkk9, mpk3, and mpk6, upon Spm treatment. The expression levels of the three bZIP genes and their target genes in mkk9, mpk3, and mpk6 were determined by qRT-PCR. The experiments were performed using three independent biological samples. The mean + SD were displayed. Asterisks indicate significant difference (∗P < 0.05 and ∗∗P < 0.01).
Spm Activates Three Pivotal bZIP Genes and their Target Genes in UPR
Previous research showed that exogenous Spm induces the expression of Arabidopsis bZIP60 and its ortholog, NtbZIP60, of N. tabacum (Tateda et al., 2008; Mitsuya et al., 2009). We addressed whether Spm induces bZIP60 specifically or rather induces the whole UPR process. Here, we showed that Spm induces not only bZIP60 but also bZIP17 and bZIP28 expression (Figures 1 and 4). In the case of bZIP60, an IRE1-dependent bZIP60 splicing process was also activated by Spm (Figures 2 and 3). We speculate that the proteolytic bZIP17 and bZIP28 processing by S1P and S2P is activated by Spm because their downstream target genes such as HB-7, RD20, CNX1, and CRT2 were concomitantly upregulated (Figure 1, data not shown). In addition, in Spm-enriched Arabidopsis plants, the three bZIP genes were induced (Figure 4). We further showed that Spm can recruit bZIP17 and bZIP60 proteins to nuclei in plant cells (Figure 5). Taken together, we conclude that Spm is a novel inducer of UPR.
Spm-Induced UPR Pathway is Mediated by the MKK9-MPK3/MPK6 Cascade
Previous work showed that Spm activates two kinds of MAPKs, SIPK and WIPK, and this activation requires the production of reactive oxygen species and Ca2+ influx in tobacco leaves (Takahashi et al., 2003, 2004). Our result showed that MKK9-MPK3/MPK6 is the MAPK cascade for Spm-induced UPR in Arabidopsis. The Arabidopsis MAPK signaling pathways are involved in various biological processes (Colcombet and Hirt, 2008; Rodriguez et al., 2010). The MKK9-MPK3/MPK6 cascade was used in ethylene signaling (Yoo et al., 2008) and was also involved in the phytoalexin production, and in salt stress response in Arabidopsis (Xu et al., 2008). Furthermore, our result showed that, in mkk9 mutant, Spm-induced bZIP17, bZIP28, and bZIP60 expression was significantly alleviated (Figure 8). Moreover, the Spm-induced expression of the respective target genes, HB-7, CNX1, and BiP3, was also abrogated in the mkk9 mutant (Figure 8). The result suggests that Spm-evoked Ca2+-elevation activates the MKK9-MPK3/MPK6 cascade, which triggers the bZIP17, bZIP28, and bZIP60 induction. It should be noted that, in mammalian system, IRE1 activates JNK protein kinases (Urano et al., 2000). To know whether Arabidopsis IRE1 can activate the MKK9-MPK3/MPK6 pathway is of interest.
How does Spm Induce UPR?
We noticed that the Spm-induced expression profile of three UPR key bZIP genes and their target genes differs from the one induced by the typical UPR inducers, DTT and TM. Martinez and Chrispeels (2003) described that the respective UPR agents have different effects; i.e., the genes induced by TM and DTT were different in Arabidopsis. It is quite reasonable because TM affects N-glycosylation whereas DTT changes the cellular redox state (Liu and Howell, 2010). Then the question arose how Spm induces the UPR. Spm-induced expression of bZIP17, bZIP28, and bZIP60 genes was blocked by a Ca2+ channel blocker (Figure 6). Spm- and T-Spm-deficient Arabidopsis plants cannot grow well in Ca2+-depleted MS medium (Yamaguchi et al., 2006), suggesting that the tetraamines are involved in Ca2+ dynamics and homeostasis. PAs are known to modulate Ca2+ dynamics via control of cation channel activity (Dobrovinskaya et al., 2000; Pottosin and Shabala, 2014). Furthermore, the action potential to regulate cation channels is the order of Spm > Spd ≥ Put, which may explain the PA specificity to evoke UPR induction. As exogenously applied T-Spm also induces Spm-responsive genes at the higher degree compared to Spm (Sagor et al., 2012b), T-Spm may possess a similar or the higher action potential in terms of modulating cation channels in relative to Spm. It is known that thapsigargin, another UPR inducer, affects Ca2+ homeostatic balance. Thapsigargin leads to ER Ca2+ depletion due to inhibition of the Sarco/ER Ca2+ ATPase (SERCA; Xu et al., 2008). Taken together, we hypothesize that Spm induces UPR pathway through affecting Ca2+ dynamics. Of course further study is needed to substantiate the hypothesis.
Physiological Relevance of Spm-Induced UPR
It was demonstrated that the polyamine Spm, exogenously applied or endogenously enriched, has a protective role in the defense response of plants against both abiotic and biotic stresses like salt, drought and heat stresses, and attack by viral pathogens (Yamaguchi et al., 2006, 2007; Mitsuya et al., 2009; Sagor et al., 2012a; Tiburcio et al., 2014; Berberich et al., 2015). A protective role was also shown for components of the UPR. When the bZIP60 ortholog of N. benthamiana was silenced by a virus-induced gene silencing approach, the host plant became hypersensitive to non-host bacterial pathogen (Tateda et al., 2008). bZIP60 was upregulated when the avirulent CMV-Y strain, but not the virulent CMV-B2 strain, was used to infect Arabidopsis ecotype C24 which carries the resistance gene, RCY1, to CMV-Y (Takahashi et al., 1994; Mitsuya et al., 2009). In parallel Spm synthase gene was upregulated and Spm content increased in the C24-CMV-Y pathosystem (Mitsuya et al., 2009). We assume a link inasmuch as the accumulated Spm activates UPR. The protection against viral infection could be accomplished indirectly by degradation of the excess Spm by polyamine oxidases which produce H2O2. H2O2 would then be transmitted to the surrounding tissue of the infection site, thereby inducing the UPR in such cells. Since the virus is unable to replicate properly while UPR takes place the tissue is protected. A distinct role of UPR in plant immunity mediated by IRE1/bZIP60 has been shown by Moreno et al. (2012). Those authors demonstrated that SA, an important phytohormone in immune response, can induce IRE1/bZIP60-mediated UPR. Nagashima et al. (2014) further showed that SA activates two signaling arms, namely bZIP28 and bZIP60, of the UPR in Arabidopsis. Those results suggest that bZIP60 (or its ortholog) has a defensive role against avirulent pathogens. In contrast, bZIP17 and bZIP28 were found to be rather associated with abiotic stresses such as drought, ABA and high salinity, and heat stress, respectively (Soderman et al., 1996; Liu et al., 2007b; Liu and Howell, 2010; Howell, 2013), for which a defensive role of Spm has also been demonstrated as mentioned above. In conclusion, the broad effect of Spm in the stress response of plants could originate, at least partly, in the capacity of Spm as an UPR inducer.
This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) to TK (26⋅04081, 15K14705) and to DK (25⋅5682). SGHM is a recipient of JSPS foreign researcher fellowship.
Conflict of Interest Statement
The Guest Associate Editor Taku Takahashi declares that, despite having previously collaborated with the authors Masaru Niitsu and Tomonobu Kusano, the review process was handled objectively. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Drs N.-H. Chua and D. Ren and Arabidopsis Biological Resource Center for providing a GFP plasmid, Atmkk9, Atmpk3, and Atmpk6 mutant seeds, respectively. Dr. Stephen H. Howell is also acknowledged for providing ire1a, ire1b, and bzip60 mutant seeds.
Supplementary Material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/article/10.3389/fpls.2015.00687
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Keywords: calcium, mitogen activated protein kinase cascade, polyamine, spermine, unfolded protein response, UPR inducer
Citation: Sagor GHM, Chawla P, Kim DW, Berberich T, Kojima S, Niitsu M and Kusano T (2015) The polyamine spermine induces the unfolded protein response via the MAPK cascade in Arabidopsis. Front. Plant Sci. 6:687. doi: 10.3389/fpls.2015.00687
Received: 20 July 2015; Accepted: 20 August 2015;
Published: 10 September 2015.
Edited by:Taku Takahashi, Okayama University, Japan
Reviewed by:Haitao Shi, Hainan University, China
Yuji Iwata, Osaka Prefecture University, Japan
Copyright © 2015 Sagor, Chawla, Kim, Berberich, Kojima, Niitsu and Kusano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Tomonobu Kusano, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai 980-8577, Japan, email@example.com
†Present address: Dong W. Kim, RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-Ku, Yokohama, Kanagawa 230-0045, Japan
‡ These authors have contributed equally to this work.


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Claire Denis
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It's released in multiple formats. All of the disc editions feature the behind-the-scene documentary Peter Jackson Invites You to the Set, the featurettes "New Zealand: Home of Middle-earth, Part 2," "Introduction to Pick-Ups Shooting," "Recap of Pick-Ups, Part 1," "Recap of Pick-Ups, Part 2? and "Music Scoring," and "Live Event: In the Cutting Room," a recording of the March 2013 Q&A and studio tour hosted by Jackson and streamed live in the web.
Bastards (IFC, DVD, Digital HD) may be the bleakest drama yet from Claire Denis, a filmmaker with her share of dark portraits. Vincent Lindon is a cargo ship captain who returns home after decades for reasons that don't become clear until much later. His sister is mess and his niece (her daughter) in the hospital, the victim of terrible sexual abuse. Their story comes together slowly in fractured flashbacks as we struggle to understand how everyone is connected, including the woman next door (Chiara Mastroianni), divorced from a powerful businessman with some shady business. What comes together most clearly is the rot in the family line and reason Kindon left it all behind. In some ways it's like a warped version of Chinatown with an even more black-hearted backstory and insidious ending. This is a tough one, but it's worth the investment. The score by Tindersticks adds a haunting atmosphere to the journey. French with English subtitles.
Jia Zhangke's A Touch of Sin (Kino Lorber, Blu-ray, DVD, Digital HD, VOD) takes four true stories of life in the modern China economy and weaves them into an unsettling portrait of the country, where the runaway growth takes its toll on the citizens racing to simply survive. Jia isn't known for a sense of humor and this film, with its stories cleverly woven together in subtle ways, has a mercenary edge to it—there's murder, predation, and bureaucratic indifference to the ordeals of citizens just trying to get by—but there's a dark humor to its satire as well. Welcome to the modern economy where everything is for sale and human capital is just another commodity. Mandarin and Cantonese with English subtitles.
'Bastards': Secrets and Surveillance from Claire Denis
If Alfred Hitchcock were still alive and exploring 21st-century modes of moviemaking, would he come up with something like Bastards? The Master of Suspense changed with the times, and maybe it's not too far-fetched to imagine him experimenting in the style operating here: a terse, elliptical, and ultimately horrifying method that withholds as much information as it doles out.
This thought passed through my mind halfway through Bastards, but make no mistake: This movie is definitely the work of French filmmaker Claire Denis (White Material, 35 Shots of Rum, Beau Travail, etc.), whose cryptic approach only adds to the film's creeping sense of unease. The picture begins by contemplating a wall of rain, as though preparing us for how hard it will be to see and understand what's going on.
Exquisite Delay: The 54th San Francisco International Film Festival + Tindersticks perform Claire Denis film scores
While Cannes assumes its privileged position in the cinematic cosmos, the extant film world lurks in relative shadow, an eclisse that nonetheless calls attention to more modestly proportioned proceedings. Still flashy in its own west coast (relaxed) way, the recently wrapped San Francisco International Film Festival – 54 and counting! – soldiered on in relatively familiar fashion and hit a sweet peak with an inspired musical program that would be the envy of any croisette flaneur.
British ensemble Tindersticks play chamber pop that lends itself favorably to the Festival's continually popular, if occasionally enigmatic, pairing of musicians with silent films at the city's choicest venue, the historic Castro theatre (note to John Waters: no cuts in the ticket line!). Straying from the usual script, the Festival enlisted the band to perform pieces from their fruitful and cryptic collaboration with director Claire Denis, (a SFIFF regular) with whom the band, in various incarnations, has worked since Denis' dreamy family drama Nenette et Boni (1996). 'Worked' is the operative term here, but it hardly conveys the depth of their engagement, so effectively have the band insinuated themselves into the textures of Denis' radically dynamic oeuvre as to become generative of it. Along with cinematographer Agnès Godard, who has acutely penetrated and exposed the supple surface of Denis' troubled world – from the latent desire lurking in bed sheets to the violence of dreamed dogs thrashing in snow on other continents – the band issue haunting and wistful treatments of Denis' often opaque narratives in unsuspecting ways, discreetly eddying around already elliptical occasions (better to think of Denis' stories in terms of thrust rather than plot) with repeated motifs tinkered out on vibes or plundered with bass, always restrained until loosed like the prevailing animal instincts onscreen.
Expecting a concert, then, would inevitably mislead. Here was the band playing song sketches to a tightly (and cleverly) edited sequence of passages – from what will likely become known as Denis' 'midcareer' – with the practicality of musical instrumentation dictating a non-chronological approach, and favoring no one film. Langour and crisis intertwine like love and hate in Denis' films – slightly indulged in Vendredi Soir, exaggeratedly in Trouble Every Day – and it's curious to listen how the band fold this in to their compositions: a flute somehow signifying a portentous note to White Material's imminent colonial collapse, a melodica riff bringing a sense of simultaneous levity and melancholy to 35 Rhums' becalmed generational divide. Propulsion and stasis are key too, as a kinetic rhythm accompanies the film's train montages, while apartment scenes are charmed with a childlike keyboard/melodica sigh, evocative of Denis' Ozu homage while capturing some of Mati Diop's beauty. With an unsensational sensitivity, the band converge the two strains like parallel tracks meeting, just as Denis manages to convey a sense of time passing both gently and tragically (Josephine's romance and Rene's death as versions of equally inevitable departure/loss).
White Material – DVD of the Week
White Material (Criterion)
Claire Denis's debut feature, Chocolate (1988), took on the legacy of French colonialism in the West African country of Cameroon through the eyes of a young French woman recalling her childhood growing up in the tensions of race, class and dislocation. Thirty years later she returned for White Material, which takes on many of the same issues from an older, more experienced perspective, both in terms of the artist and our protagonist.
Isabelle Huppert plays Maria Vial, French by ancestry, African by birth. Denis was raised in Cameroon until the age of 13 and the experience still clearly haunts her, but Maria is no stand-in for Denis. Maria is a woman in an unnamed West African trying to hold on to her family coffee plantation that her family no longer cares about while a civil war rages around her.
The film opens in the midst of chaos and fear as rebels advance on this dusty patch of country and Maria defies the tide of evacuation to return to her farm. Huppert's incarnation of the intensity and will of Maria, beyond logic or safety, powers the film. She is maddeningly single-minded, risking not just her life but her family and the day workers she rounds up to help harvest the crop when her employees run off. She refuses to acknowledge the danger and hides the truth of the situation from everyone else. Meanwhile armed child soldiers wander the property, looting the "white material" of European habitation, and rebels close in as one rebel leader (Isaach de Bankolé) bleeds out in a corner of the plantation.

subset: commoncrawl


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The Debt Recovery and Fraud Identification Solution for Local Authorities
The most comprehensive data hub designed exclusively for Local Authorities to increase revenues and improve efficiency in tracing debtors and tackling fraud
What is LoCTA?
Established in 2006, LoCTA is the first search hub to share and integrate Local Authority and national data sets, creating the broadest and most comprehensive data hub available to the public sector.
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Updated weekly, the LoCTA Hub features a range of flexible searches and filters to ensure accuracy, reduce false positives and increase hit rates.
Where is LoCTA used?
One license can be used across all Local Authority departments enabling effective data sharing and driving cost and time efficiency.
Recent legislation changes now enables any department within a Local Authority to use the LoCTA Hub subject to their own internal GDPR compliance procedures. Should any data need to be witheld the LoCTA Hub provides the facility for users to do so.
- Unlimited Usage
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Original and most trusted data hub providing secure tracing and fraud detection solutions for the Public Sector since 2006.
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Built-in filters for SBRR / SBBS and mixed hereditament
Generates SBRR / SBBS Alerts (weekly report)
Further datasets being incorporated
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"After a year of using TransUnion via our LoCTA Hub Licence, we have reduced our Credit Reference searches by 26% and the cost by 35%. We've achieved this via the exclusive 4 for 1 searches that MAG:NET secured with TransUnion. The users and contracts are all maintained by our Account Manager at MAG:NET, which means we can roll all of our tracing needs into one easy contract."
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A major trucking body has said no changes to freight regulations are required after a shocking explosion injured eight people on an outback Queensland highway.
A truck carrying ammonium nitrate rolled between Cunnamulla and Charleville rolled and exploded on Friday, destroying part of the Mitchell Highway and two bridges.
Ammonium nitrate is strictly regulated by the Department of Natural Resources and Mines but it is permitted for transportation by truck.
Queensland Trucking Association Peter Garske said the tough controls already in place for transporting explosives don't need to be updated in the wake of the blast, the Brisbane Times has reported.
"It is highly regulated in terms of the routes they can carry, in terms of the standards of training and accreditation that the drivers must have and it is highly regulated in terms of the standards of the vehicles they must use," he said.
He said using rail lines would be too inefficient for useful transport of explosives, and it would cost too much to make trains a viable freight alternative to trucks.
Queensland's Transport Minister Scott Emerson said the department will investigate the incident, but that there would "always be a role for the trucking industry in moving freight around Queensland".
The government's own Moving Freight strategy recommends increasing the amount of cargo moved by rail.
The 33-year-old truck driver remains in a critical condition after the explosion.


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1) When your opponent plays a powerful ally that you want to get rid of, 5AP is usually more than enough to do so.
2) When your opponent has no allies to defend with, attack with a 5AP Pyro for an almost guaranteed 5 direct damage.
Strong against: slow-developing and quality decks, decks with low ally count.
Weak against: direct damage, especially Shoot a Fireball, ally-heavy decks.
Community content is available under CC-BY-SA unless otherwise noted.


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The Samsung Omnia M, that Samsung Smartphone that runs on Windows Phone announced back in May was announced in India and is said to be available in the UK in August 1. For the Indian market, the smartphone's product page is now up and Samsung gave the device a price of Rs 16,400 which can be bought right from the company's website. In the UK, on the other hand, Phones4U is said to be offering the device 2months before any other retailer would in that specific market. Phones4U offers the device, SIM-free, for £289.95 but you can also get it tied up with carriers.
July 31, 2012
India and the UK gets the Samsung Omnia M
Labels: Availability , Cellphones , IND , News , Phones4U , Pricing , Samsung , Samsung Omnia M , UK , Windows Phone
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N30 Strike
Best wishes to everyone who's on strike tomorrow as the British working class flexes its (somewhat atrophied) muscles in what promises to be the largest day of industrial action the country has seen during my lifetime. As a UCU member I'll be on strike and picketing from early (to paraphrase a football chant, 'We can see you sneaking in'), then I'm hoping to meet the GMB march from Cheltenham General Hospital when they arrive at the Municipal Offices about 10am, and, after that, I'll be heading with some colleagues to the march and rally in Gloucester (meet 1pm at Shire hall). By that time, I'll be due refreshments.
The Day of Action is supported by an unprecedented number of Trade Unions including AEP, ASPECT, AHDS, ATL, CSP, EIS, FDA, GMB, NAHT, NAPO, NIPSA, NUT, PCS, Prospect, RMT (Royal Fleet Auxiliary), SCP, SIPTU, SoR, TSSA, UCAC, UCATT, UCU, Unison, and Unite. I don't even know who half of them are. For a sample of what's going on around the country, check here.
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On This Page: | Ticket Info | Trailer | Summary | About the Filmmaker, Cast & Crew |
| Interview with the Director | The Opera in Diva | Dinner and a Movie |
Screening at 7:00 pm,
doors open at 6:30 pm
953 Eden Park Drive, Eden Park/Mt. Adams.
click for Directions & Map
$7 tickets are ONLY available online, by phone, at the Museum, and at the door subject to availability.
...and at these locations
($9 tix only, cash only),
click each location below for a map:
Sitwell's Coffee House
513 281 7487
Lookout Joe Coffee Roasters
513 871 8626
Shake It Music & Video
513 591 0123
Coffee Emporium
513 651 5483
Tickets will also be available at the door, subject to availability.
To complete your night out, we've arranged discounts with two restaurants offering great food at reasonable prices:
Wednesday, January 30,
Vito's Cafe. Located 8-10 minutes from the Art Museum at 654 Highland Avenue, Ft. Thomas, KY.
CWC patrons attending DIVA on Wednesday night will receive a complimentary glass of wine ($6-10 value) before the film. Vito's is closed on Tuesdays, but if you attend the Tuesday film, simply take your ticket stub and dine on Wednesday.
Vito and Mary run an Italian-style neighborhood trattoria, featuring great soups, pasta and veal, chicken, beef and vegetarian entrees. Vito's Cafe is the home of the singing servers - you'll enjoy light opera and contemporary songs along with a great meal.
Read about Vito's Cafe in this Enquirer review. Reservations suggested, call 859.442.9444. Click here for directions, menu and general info .
Wednesday, January 30, The Terrace Cafe at the Cincinnati Art Museum. Non-members attending LUNAFEST on this night will receive the same 10% discount (excluding alcohol) extended to museum members. The restaurant fills up quickly on film nights, so reservations are recommended, call 513.639.2986. View menu here.
"A top-drawer French thriller, with romance, comedy, opera and murder! Gorgeous visuals, sound and music, one-of-a-kind characters, a sheer joy to watch!"
"One of the best thrillers of recent years, but more than that, a brilliant film and visual extravaganza that announces the considerable gifts of director Jean-Jacques Beineix."
Read the CityBeat review of Diva by Steve Rosen. (Also available on page 43 of the January 23-39 hardcopy edition.)
1982 saw the American release of the film that launched a second New Wave, the cinéma du look, of French cinema. DIVA, the debut creation of director Jean-Jacques Beineix, won four Césars (French Oscars) including Best First Film, Cinematography, Music and Sound.
An art-house sensation, DIVA, played for over a year in some cinemas, but had only brief exposure in Cincinnati. Now, with a new 35mm print, sound track, translation and subtitles, you can enjoy this unique and exciting thriller, full of comedy, romance, opera and murder!
Diva single-handedly launched the Cinéma du look, an explosion of visually stunning, punk-inspired, super-cool French movies in the early 80s.
"The Cinéma du look was a French film movement of the 1980s. It referred to films that had a slick visual style and a focus on young, alienated characters that were said to represent the marginalized youth of Francois Mitterrand's France. The three main directors of the Cinéma du look were Jean-Jacques Beineix, Luc Besson and Leos Carax. Themes that run through many of their films include doomed love affairs, young people with peer groups rather than families, a cynical view of the police and the use of the Paris Métro to symbolize an alternative, underground society. The mixture of 'high' culture, such as the opera music of Diva and Les Amants du Pont-Neuf and pop culture, for example the references to Batman in Subway, was another key feature. Unlike most film movements, the Cinéma du look had no clear political ideology. -Wikipedia
Ginette Vincendeau, in her Companion to French Cinema (Cassell, 1996), defines the films made by Beineix, Besson and Carax as "youth oriented films with high production values… The "look" of the Cinéma du look refers to the films' high investment in non-naturalistic, self-conscious aesthetics, notably intense colors and lighting effects. Their spectacular (studio-based) and technically brilliant mise-en-scène is usually put to the service of romantic plots."
"Most of Beineix's feature films to date were released in a single decade, the 1980s, and he is generally seen as the best example of what came to be known as the Cinéma du look. This was one of the two new types of film to emerge in the 80s (the other being the heritage film), to join the other popular French genres of the comedy and the policier. The Cinéma du look was placed by many, including Beineix himself, in a position of confrontation with the cinema of the Nouvelle Vague - the New Wave. Just as Truffaut had famously attacked the filmmakers of the 1950's, the tradition de qualité, so did Beineix thirty years later by attacking the Nouvelle Vague and the establishment of critics who supported the modernist cinema it represented, for being out of touch with contemporary, and especially young, audiences." - Phil Powrie, Jean-Jacques Beineix (Manchester University Press, 2001)
Filmmaker, Crew & Cast
JEAN-JACQUES BEINEIX (Writer/Director)
Born in Paris on October 8, 1946, Beineix developed an interest in cinema at a young age. After discovering the medium through repeated viewings of old 16mm films at a local film club, he began making 8mm shorts with his friends when he was 16. During the 70s, he gave up studying medicine to start his career in film.
He became an established assistant director, working with Claude Berri, René Clément, Claude Zidi, Jean-Louis Trintignant and even Jerry Lewis (on the ill-fated The Day the Clown Cried). But, like many assistants, Beineix's ultimate dream was to direct. He had a chance in 1977 with the short film Le Chien de Monsieur Michel. A promising debut, it won the first prize at the Trouville Film Festival and earned a César nomination for Best Short Film (fiction).
In 1981 came Diva, which became an international hit, playing for over a year in some cinemas. Next came the expensive The Moon in the Gutter. Adapted from a David Goodis novel, the film was even more radical than Diva in its deliberate artificiality. The film was booed at its premiered at the Cannes Film Festival in 1983. Only few voices rose up to defend the movie but it was not enough to save it. It flopped at the box office but won a César award for Best Production Design.
Beineix bounced back in force in 1986 with 37°2 le matin (Betty Blue), based on a Philippe Djian novel. The film was another international hit, won the top prize at Montréal Film Festival, and was nominated for Best Foreign Film at both the Oscars and Golden Globes. It also earned 9 César nominations including Best Film and Best Director.
Beineix's next movie Roselyne et les lions (1989) was set in the circus world. In 1992, IP5 featured Yves Montand's last role. Beineix then resurfaced with social documentaries. He did a film about children in Romania; Otaku was shot in Japan; and Assigné à résidence was about locked-in syndrome victim Jean-Dominique Bauby, the subject of Julian Schnabel's recently released film The Diving Bell and the Butterfly.
In 2001, he returned to fiction with Mortel Transfert, a psychological thriller based on a Jean-Pierre Gattégno novel. In 2002, Beineix drew strong ratings with made-for-TV documentary Loft Paradox, an attempt to analyze the success of French reality show Loft Story.
With his intense focus on the power of images, Beineix paved the way for directors like Luc Besson, Leos Carax and Jean-Pierre Jeunet. With Diva and 37°2 le matin, he directed two of the few French films of the 80s that reached an international audience.
PHILIPPE ROUSSELOT (Cinematographer)
Among the most influential cinematographers of our time, Philippe Rousselot assisted Nestor Almendros on three Eric Rohmer films before graduating to director of photography in 1972. Rousselot had already enjoyed fruitful collaborations with directors Diane Kurys and Claude Goretta before landing in the spotlight with Jean-Jacques Beineix' s Diva. The film won him his first César award, as well as the Best Cinematography Award from the National Society of Film Critics, and kicked off a prestigious international career.
Rousselot won a second César for Alain Cavalier's Therese (1986) around the time he began venturing into English-language cinema, evoking the passions of the 18th century French aristocracy in Dangerous Liaisons (1988, ASC nomination for Best Cinematography) and those of 20th century literati Henry Miller and Anais Nin in Henry and June (1990, Oscar-nominated).
Rousselot has often worked with John Boorman, including on Hope and Glory (1987), which brought him a second Academy nomination and the British Society of Cinematographers award, and The Emerald Forest (1985). Woodlands have provided a showcase for some of Rousselot's finest work -- A River Runs Through It (1992), for which he earned a Best Cinematography Oscar, and The Bear (1988, ASC nomination), for which he built a number of camera mounts and other devices to ingeniously mimic a bear's-eye view of the world.
Rousselot returned to France to shoot the Patrice Chéreau's Queen Margot (1994), which earned him a third César. He subsequently provided the burnished tones for Neil Jordan's Interview with the Vampire (also 1994, winner of both the BAFTA and British Society of Cinematographer's award) and Stephen Frears' Mary Reilly (1996). In 1997, he stepped behind the camera to direct The Serpent's Kiss.
In the past few years he collaborated with Tim Burton -- Planet of the Apes, Big Fish and Charlie and the Chocolate Factory. He is the cinematographer of The Brave One, directed by Neil Jordan and starring Jodie Foster, currently in theatres.
HILTON McCONNICO (Production Designer)
Born in Memphis, Tennessee in 1943, designer and artist Hilton McConnico has lived and worked in Paris since 1965 and has since established himself as one of the world's most accomplished designers.
After working in fashion for such designers as Ted Lapidus, Yves St. Laurent, and Balenciaga, he was set designer and art director for more than 20 films. He collaborated with Beineix on Diva and on 1983's The Moon in the Gutter, which earned him a César award for Best Production Design. He was also the production designer on Truffaut's final movie, Confidentially Yours! He then moved to the other side of the camera to produce some 30 commercials and video clips.
His collaboration with Daum Crystal began in 1987; some of his "Cactus" creations for the manufacturer were presented by former French President François Mitterrand to President George H. W. Bush as a gift of state. He was also the first American to have work permanently inducted into the Louvre's Decorative Arts collection. His "Cactus" creations and the "Pepper Carpets" of Toulemonde Bouchart are included in permanent collections throughout the world, including the decorative arts Museums of Paris, New York, Oslo and Lausanne.
McConnico continues to be active on the global design scene, especially in architecture and interior design. Recent projects include the Toupary restaurant on the fifth-floor of the historic Samaritaine department store in Paris and the Hermes Museum in Tokyo, which he conceived for the new Renzo Piano building in the famed Ginza shopping district. In 2005, the Sommet du luxe et de la creation (International luxury and design exhibition) recognized McConnico for his entire body of work with the Talent de L'Audace award. The January 2006 Scènes d'interiéur interior design exhibition paid him tribute in a retrospective created by McConnico himself.
VLADIMIR COSMA (Composer)
One of France and Europe's most distinguished film composers, Vladimir Cosma has scored more than 150 films and TV productions. Though he enjoyed almost immediate success in comedies, he continued experimenting with different styles and genres, and this versatility brought him international acclaim.
Born in 1940 in Bucharest, Romania, the son of a renowned conductor and concert pianist, Cosma studied music from his early years onward, eventually attending the National Conservatory in Bucharest (from which he graduated with two first prizes, for violin and composition). In 1963 he went to Paris to advance his studies at the French Conservatory, where, in addition to his classical background, he developed an interest in jazz, folk music, and film music. Between 1964 and 1967, he toured the world as a concert violinist, visiting the U.S., Latin America, and Southeast Asia. A meeting with popular film composer Michel Legrand became the first step towards his future career. Although Cosma always mentions Legrand's importance, he also admits the influence of such composers as Burt Bacharach and Henri Mancini.
In 1967, Cosma began his long-running partnership with film director Yves Robert for whom he scored the international hits The Tall Blond One with One Back Shoe (1972) and its sequel, as well as Marcel Pagnol's autobiographical diptych, La Gloire de Mon Père and Le Chateau de ma Mère (both in 1990). He also wrote the music for several comedies directed by Francis Veber and Gerard Oury, starring such hit French comedians as Pierre Richard and Louis de Funčs.
One of his biggest international hits was the Satie-inspired impressionistic piano pieces for the soundtrack for Diva, for which he was awarded his first César. He received another César for Ettore Scola's Le Bal (1983), and the main instrumental theme from it became a hit worldwide. He also received a Cannes Film Festival award for the entire body of his work.
WILHELMENIA WIGGINS FERNANDEZ (Cynthia Hawkins)
The American Soprano was born Philadelphia in 1949. Ms Fernandez's early training was at the Philadelphia Academy of Vocal Arts, followed by a scholarship at the Juilliard School of Music in New York. Her operatic debut was as Bess in Porgy and Bess for the Houston Grand Opera, a production which then opened on Broadway and toured both the U.S. and Europe.
In 1979, she made her début in Paris as Musetta in La Boheme (with Placido Domingo and Kiri Te Kanawa) and sang the same role at the New York City Opera in 1982. It was while performing Musetta in Paris that she met Jean-Jacques Beineix. She recalls that he went backstage for an autograph. Then he just stood and watched. When Fernandez asked him, "Is there something else?" he asked her to read a script. "Me? In a film? I'm not an actress, I'm an opera singer", she reported. The next day she handed the script back with a refusal.
After Beineix convinced her to read the script with him, the idea grew more appealing. "I started thinking of reaching out to all those people who say they don't like opera because they don't understand it. I was hoping that I would entice more people to see opera."
Since Diva, the soprano has sung in operas and recitals in cities all over the world, her most notable roles were Carmen in Carmen Jones, for which she received the Laurence Olivier Theatre Award in 1992 as Best Actress in a Musical, and Giuseppe Verdi's Aida, a role she has performed in Luxor and at the Pyramids in Egypt.
Unlike her character in Diva, she has made recordings of George Gershwin and of Negro Spirituals.
FRÉDÉRIC ANDREI (Jules)
Born in 1959, the son of a famous veteran TV director, Andrei first appeared in films by Yves Boisset and Michel Deville before landing the role of the opera loving postman in Diva. The success of the film allowed him to direct a short the following year, then a feature film, Paris minuit, in 1986. As an actor he has been seen in Eric Heumann's Port Djema (1996) and Tonie Marshall's hit comedy, Venus Beauty Institute (1998).
RICHARD BOHRINGER (Gorodish)
Born in Moulins, France in 1942, Richard Bohringer began his film acting career in 1970 but did not make his first career breakthrough until Francois Truffaut's The Last Metro (1980), before being launched to international stardom by his role of Gorodish in Diva.
In 1985, director Luc Besson cast him in Subway, starring Isabelle Adjani and Christopher Lambert. That same year, Bohringer received a Best Supporting Actor César for his role in Denis Amar's 1984 prison melodrama L'Addition. In 1988, he received his second César, this time as Best Actor, for Jean-Loup Hubert's Le Grand Chemin. In 1989 he was The Cook in Peter Greenaway's The Cook the Thief His Wife & Her Lover.
Counting some 130 roles to date in both feature films and TV, Bohringer has notably worked Claude Miller, Patrice Leconte, Idrissa Ouedraogo, Jean-Pierre Mocky, Kim Ki-Duk, Robin Davis, Charles Matton, Peter Bogdanovich, Michel Deville, Jean-Charles Tachella, and others. In 1997, he won France's TV award Sept D'Or for his performance in the mini-series Un homme en colére.
Bohringer is the author of plays, scripts, books and travel writings, and has also directed for cinema and TV, notably the 2003 feature, C'est beau une ville la nuit, based on his 1988 book. A lover of Africa, Bohringer took Senegalese citizenship in 2002.
THUY AN LUU (Alba)
Discovered by Beineix's casting director Dominique Besnehard, the supposedly-15-year-old Thuy An Luu impressed the director so much that he rewrote the part of Lolita-like Alba, changing her from a North African to a Vietnamese girl. She subsequently appeared in a handful for films and TV productions, then dropped out of sight in the late 80s.
DOMINIQUE PINON (The Priest)
Rubber-faced French actor Dominique Pinon was born in 1955. It was casting director Dominique Besnehard who recommended him to Beineix for the role of the laconic man, The Priest, in Diva, a role that brought him to international attention and remains among his signature roles. In 1983, Pinon received a César nomination as Most Promising Actor for his performance in The Return of Martin Guerre (1982).
His unusual and pronounced features led him to play a series of secondary characters in important films. He was the alcoholic brother of Gérard Depardieu in Beineix's second feature The Moon in the Gutter (1983), a bum in Ermanno Olmi's The Legend of the Holy Drinker (1988) and a vagabond in Roman Polanski's Frantic (1988).
Diva aside, Pinon is best known for his work with Marc Caro and Jean-Pierre Jeunet. His malleable, idiosyncratic features made him seem a natural part of Caro and Jeunet's surreal landscape, as demonstrated in both Delicatessen (1991) and La Cité Des Enfants Perdus (The City of Lost Children, 1995). In 1997, he collaborated with Jeunet alone on a rare American outing, Alien Resurrection, in which he played a paralyzed mechanic opposite Sigourney "Ripley" Weaver and Wynona Ryder. In 2001 and 2005, Jeunet cast him once again in the international smash hit, Amelie, and A Very Long Engagement. In 2004, he received the Moliére Award (France's national theatre award) for his comic performance in a Roland Topor play.
GÉRARD DARMON (The Caribbean)
Born in Paris in 1948, Gérard Darmon has played a wide variety of roles in many different genres to become one of France's most popular supporting actors. His first major role was in Gérard Oury's farce, The Mad Adventures of Rabbi Jacob (1973), but it was Diva and Alexandre Arcady's Jewish gangster epic, Le Grand Pardon (1981), that brought him critical and public attention. He received his first César nomination as Best Supporting Actor in Beineix' 37°2 le matin (Betty Blue, 1986) and became a regular player in a series of Claude Lelouch films.
Darmon won his second Best Supporting Actor César nomination for Astérix et Obélix: Mission Cléopatre (2002) and co-starred opposite Nick Nolte in Neil Jordan's The Good Thief, a remake of Rialto Pictures' Bob Le Flambeur. Winner of the Jean Gabin Prize in 1983, Darmon recently branched out into music with two albums of pop songs and a couple of performances at the prestigious Olympia Music Hall in Paris.
JACQUES FABBRI (Saporta)
Distinguished French actor Jacques Fabbri was born in Paris in 1925. He was the founder of a theatrical company. In 1965, he directed A Midsummer Night's Dream at the Comédie Française and made his debut as film director with the comedy Les pieds dans le platre. In his long career as a character actor, he appeared in many plays and films, including Jacques Becker's Rendez-vous de Juillet (1949) and René Clair's Les Grandes Manoevres (1955). He died in 1997.
An Interview with Jean-Jacques Beineix
Irène Silberman called me and asked "Did you read Diva?" I said no but that I would. I read it the same day and it was incredible because I was immediately taken by the characters. By the singer, by the postman - who wasn't a postman in the novel but a delivery man for a record company - I was seduced by this character, his solitude, his passion, the fact that he is a collector. And I was seduced by this love story paired with the police intrigue and the mystery. It was a genre movie, and at the same time it touched all genres. At the time we used to make autobiographical films. This film was the opposite, it was a commissioned film but one where I could include a lot of myself without anyone realizing it, because, in fact, the postman Jules is a little bit me.
Diva tells the story of our time, the story of the artist facing the mechanism of production. It's a movie that deals with the situation of the artist faced with the means of recording. The subject matter is very modern; Diva is very, very, modern. When you see the movie again today you realize it hasn't aged.
I put in this picture everything there was. The scenery around us today is artificial, with drawings on the walls, advertisements, the décor. We live in a world of décors, we live in a fake world, and this fake world is a reality, our daily life. And Diva plays with all this, including the classic characters of the detective novel who have been diverted. It's a diversion of genres.
It's the mission of the artist to invent, to provoke, to be subversive and different. Everywhere I found conventions and archetypes in filmmaking. With 80 years of cinema behind us, I could not understand why so many movies kept saying things in the same way. I wanted to regain the pleasure of surprise that I had as a kid. While filming Diva I did everything and anything but, at the same time, with a kind of conviction, with sincerity and without distancing. I was asking incredible things from Philippe Rousselot who used to assume the lotus position to calm down, dripping with sweat. Serge Silberman used to threaten to stop filming every other day. Ully Pickard, the head of production would say "You need a crane, but will you know how to use it?" I was close to saying, "Shut up, I invented the crane!"
And then there was the wonderful meeting with Wilhelmenia, with whom I fell immediately in love and I became the postman. And there was Dominique Pinon whom I had wanted for the first movie I was supposed to do and never did - I had written a script. There it was -- a miracle!
When the movie came out, I really had the impression I had made my first and last movie because the reviews were horrendous. I was butchered. They said I had grandiose taste, that the movie cost a fortune, that there was no script. Some journalists liked it, but they didn't know what to write about it. Diva is unique. It can't be done again. The career of this film is unique. When I say that I felt I made two movies for the price of one, my first and my last one, it's because the film at first was a catastrophe. Few remember this because in the end it was seen by one million people in Paris, but the first year only one hundred thousand people saw it. It was at a stand-still after one year. Can you imagine a film today in theaters for one year with one hundred thousand viewers and after one year, boom, suddenly one million viewers in Paris alone?
I insist that it is a very modern film, but I shouldn't be saying that, I will sound pretentious. Yet luck, the novel and the characters make it a modern film, with modern actors. But it was another era. The cinema has changed since then. That's the way it is. There will be other genres, but I don't know if there will be other Divas.
The first time I saw her, she was smashing plates with incomparable authority on the immense stage of the Opéra de Paris. A black and capricious Musetta, dressed in red velvet, beautiful and violent, she already fascinated me, and then there was her voice, perfectly round and velvety, her singing perfectly sustained by the physical involvement in the part. I had found my Diva.
Many months of research, trips, and meetings: the black divas of London, Munich, and New York; a list that we thought was complete and to which there was always one more name to add. After the performance, I did not go to see her. I needed to wait for the excitement to pass, for only then I would know if the dream was reality.
It became a reality the next day, when she appeared in my non-descript office, in a red dress and an immense purple shawl. She was very simple and I suddenly found myself in the difficult situation of having to ask her to play a diva. We talked; she knew nothing about cinema, I knew nothing about opera, everything was possible, so why not shoot an opera-policier?
As I watched Wilhelmenia, I knew that she was Cynthia Hawkins, the diva, and that no one else would play that part. Cinema, shooting: the time when the exceptional meets the ordinary. She worked extremely professionally, with dignity, always on time and diligent.
Through her I understood that opera is a form of asceticism that many movie actors should turn to for inspiration. It is impossible to reduce a person to a sum of qualities and faults. I treasure the memory of a passionate woman, possessed by music, terrorized before a concert, jubilant after it, always ready to burst into flames over one note. She was Diva, and Diva was how I wanted her. I will never forget when we went to London, before the film, to record the music…her excitement, and mine, as she advanced alone, in front of the musicians of the London Orchestra, and then, finally liberated from the stage fright that oppressed her, her voice started to rise…
For that Wally, I will be forever grateful.
"When the film was finished, I asked several prominent people in the opera world to see the film, not to give me a judgment of it as a film, but to see if the music could stand up to the scrutiny of experts. They said yes, but I was still concerned if it would stand up to American opera lovers." - Beineix
La Wally is a four-act opera composed by Alfredo Catalani to a libretto by Luigi Illica and first performed at La Scala, Milan on January 20, 1892. The libretto is based on a theatre piece by Wilhelmine von Hillern (1836-1916), Die Geyer-Wally, Eine Geschichte aus den Tyroler Alpen (literally: "The Vulture-Wally"). The story is set in a village in Tyrol. The father of the heroine, Schrodinger, wants to arrange a marriage for her that she does not want. Wally instead leaves her home with the minstrel Hagenbach.
The opera is best known for its aria Ebben? Ne andró lontana, (Act I), sung when Wally decides to leave her home forever. It is a must in the repertoire and recitals of the greatest sopranos, yet before Diva it was mostly unknown to larger audiences. Cynthia Hawkins sings this aria in the recital that opens Diva, and the aria is featured prominently throughout the film. Following are the lyrics:
Ebben? Ne andró lontana,
come va l'eco della pia campana...
lá, fra la neve bianca,
lá, fra le nubi d'or...
lá dove é la speranza,
la speranza, il rimpianto, il rimpianto,
e il dolor!
O della madre mia casa gioconda
la Wally ne andrá da te,
da te, lontana assai,
e forse a te, e forse a te,
non fará mai piú ritorno, ne piú la rivedrai.
Mai piú, mai piú.
Ne andró sola e lontana,
come l'eco della pia campana...
lá, fra la neve bianca.
Ne andró, ne andró,
sola e lontana...
E fra le nubi d'or!


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required reading list
what I learned sheet
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psalm 91
For memorization prior to Black Belt test.