A Nuclear Factor Regulates Abscisic Acid Responses in Arabidopsis

Abscisic acid (ABA) is a plant hormone that regulates plant growth as well as stress responses. In this study, we identified and characterized a new Arabidopsis protein, Nuclear Protein X1 (NPX1), which was up-regulated by stress and treatment with exogenous ABA. Stomatal closure, seed germination, and primary root growth are well known ABA responses that were less sensitive to ABA in NPX1 overexpressing plants (NPX1-ox). NPX1-ox plants were more drought sensitive and the changes in response to drought were due to the altered guard cell sensitivity to ABA in transgenic plants and not due to a lack of ABA production. The nuclear localization of NPX1 correlated with changes in the expression of genes involved in ABA biosynthesis and ABA signal transduction. To understand the function of NPX1 we searched for interacting proteins and found an ABA-inducible NAC transcription factor, TIP, interacted with NPX1. Based on the whole plant phenotypes we hypothesized that NPX1 acts as a transcriptional repressor and this was demonstrated in yeast where we showed that TIP was repressed by NPX1. Our results indicate that the previously unknown protein NPX1 acts as a negative regulator in plant response to changes in environmental conditions through the control of ABA regulated gene expression. The characterization of this factor enhances our understanding of guard cell function and the mechanisms that plants use to modulate water loss from leaves under drought conditions. at t-test).


INTRODUCTION
In plant and animal genomes there is a relatively high percentage of unknown genes that currently lack defined motifs or domains (Gollery et al., 2006). Most of the differences in the composition of genes between species have been attributed to proteins with obscure features (POF). Arabidopsis contains 5069 POFs and 2045 have no obvious homologs in rice or poplar. These proteins with obscure features have been suggested to be involved in species-or phylogenetic-specific functions in Arabidopsis. The study of unknown genes or POFs is therefore an important task which should provide information that will enhance our understanding of what makes plant species unique (Gollery et al., 2007). A couple of approaches have been suggested for defining the function of proteins of unknown functions. Genome-wide clustering and the analysis of gene function in clusters allowed for the association of 1,541 proteins of unknown function with coexpressed genes for proteins of known function (Horan et al., 2008). Gene expression analysis was also used to identify a comprehensive set of abiotic stress-response genes (Horan et al., 2008). Coexpression and genome wide transcript profiling data represents an important resource for guiding future functional characterization experiments of proteins or genes of unknown function. In this study we were able to develop hypotheses as to potential gene function based on available expression data in AtGenExpress Database (Arabidopsis eFP Browser) (Winter et al., 2007).
Plants grow in environments where they encounter dynamic stresses such as cold, drought and variations in nutrient concentrations. Molecular and cellular responses to abiotic stresses are becoming understood which will allow for the engineering of how plants respond to such conditions (Zhu, 2002;Shinozaki and Yamaguchi-Shinozaki, 2007). The early events of plant adaptation to environmental stresses triggers specific and more general stress signal transduction networks leading to specific changes in gene expression. The up-regulation of expression of several stress-regulated genes results in improved tolerance to drought, salt and cold (Kasuga et al., 1999;Saijo et al., 2000).
The importance of potassium for plant growth and development is well documented. It plays a role in a wide range of functions in plants including photosynthesis, enzyme activation, protein synthesis, osmotic adjustment (Marschner, 1995), and transpiration (Schroeder et al., 2001). The protein of unknown function that we characterized in this work was identified as being regulated by potassium availability.
Potassium is also known to be important under drought conditions. Stomatal closure www.plantphysiol.org on September 3, 2017 -Published by Downloaded from Copyright © 2009 American Society of Plant Biologists. All rights reserved.
occurs following K + and anion efflux, resulting in the loss of water from the cell leading to a reduction in cell turgor and pore closure (Schroeder et al., 2001). Reducing water loss through stomatal closure is one strategy to minimize the adverse effects of drought (Marta Riera et al., 2005) and therefore K + homeostasis may be considered as a key factor in drought adaptation.
The phytohormone abscisic acid (ABA) is also a key factor in regulating developmental and physiological processes in plants, including seed dormancy and germination and seedling growth, as well as in controlling responses to many abiotic stresses such as drought (Schroeder et al., 2001;Assmann, 2003;Yamaguchi-Shinozaki and Shinozaki, 2006). Under drought conditions, the ABA concentrations in leaves increase and this increased ABA acts as a signal (Christmann et al., 2007;Schachtman and Goodger, 2008). The production of ABA in roots and its transport to the leaves also provides the plant with a mechanism for transmitting a signal to report on the water status of the soil. ABA is also required to maintain root growth in addition to its role in reducing transpiration by triggering stomatal closure under water deficit (Schachtman and Goodger, 2008). Additionally, ABA may act as a signal under reduced nutrient supply (Peuke AD et al., 1994;Peuke et al., 2002). Although increased ABA in grains from K + -deficient wheat plants and increased ABA concentrations in K + -deficient wheat flag leaves (Haeder and Beringer, 1981) have been measured the physiological consequences of this increase in ABA are unknown.
To identify the initial cellular responses to K + deprivation, we performed microarray experiments on Arabidopsis roots (Shin and Schachtman, 2004

Expression of NPX1 Was Enhanced Under Stress Conditions
Previously, we found an unknown Arabidopsis nuclear factor Nuclear Protein X1 (NPX1) was up-regulated in roots under K + deprivation. To determine whether NPX1 is regulated by other nutrient stresses, real-time PCR was performed on root RNA isolated from plants that had been deprived of potassium, nitrogen or phosphorus. The abundance of the NPX1 transcript increased significantly in roots starved of K + for 6 h (Fig. 1A). NPX1 was widely expressed in different tissues (Supplemental Fig. S1) and also found to be regulated by abiotic stresses as determined by the AtGenExpress Database (Arabidopsis eFP Browser) (Winter et al., 2007). We verified that the expression of NPX1 increased in response to salt stress ( Fig. 1B), increased under cold stress ( Fig.   1C) and responded to ABA treatment (Fig. 1D). The NPX1 gene expression patterns suggested that NPX1 may be involved in ABA dependent abiotic stress signaling.

Overexpression of NPX1 Causes ABA Insensitivity
To further elucidate the function of NPX1, a homozygous T-DNA inactivation line npx1-1  S2). The growth of npx1-1 was tested multiple times and we found no significant differences in growth as compared to the wild type under control or K + deprived conditions.
To elucidate the role of NPX1 in ABA signaling, we tested whether the disruption or overexpression of NPX1 affected ABA responses. Since ABA signaling is known to be an important component in seed germination, we determined the ABA sensitivity of germination ( Fig. 2A). In the absence of exogenous ABA, NPX1-ox and npx1-1 mutant seeds germinated as well as wild-type seeds ( Fig. 2A); and in the presence of 0.3 µM and 0.5 µM ABA, NPX1-ox seed germination was less sensitive to ABA whereas npx1-1 seed germination was more sensitive. NYFP-NPX1-ox seed germination was also less sensitive to ABA treatment (Supplemental Fig. S2B). After germination ABA may still regulate seedling growth; and therefore, we analyzed this aspect of ABA sensitivity by transferring 6-d-old seedlings on 0.25x Murashige and Skoog (MS) with sufficient potassium to plates containing 0, 3, 10, 20, and 50 µM ABA (Kuhn et al., 2006). The primary root of NPX1-ox and two NYFP-NPX1-ox were longer than the wild type under control conditions and also on medium containing 3 to 50 µM ABA ( Fig. 2B and Supplemental Fig. S2C) whereas the primary root growth of npx1-1 was more sensitive to ABA. The complemented NPX1-transformed npx1-1 plants (npx1-1-NPX1) showed the same ABA sensitivity as the wild type in germination and root growth (Supplemental Fig. S2). When grown under K + deprived conditions, the primary root length of NPX1-ox was significantly longer as compared to wild type (Supplemental Fig. S3).
Since it is well known that both ABA and potassium are critical components in stomatal movement and response to drought, we analyzed stomatal responses to ABA in loss-and gain-of-function NPX1 plants (Fig. 2C). Compared to wild type, guard cells from NPX1-ox plants exhibited greater insensitivity to ABA-induced stomatal closure.
Ten micromolar ABA (Fig. 2C) resulted in stomatal closure in wild-type plants, but not in the NPX1-ox lines. In contrast, the disruption of NPX1 increased stomatal sensitivity and stomatal closure responses at 1 µM. These data show that NPX1 plays an important role in multiple processes that involve ABA signal transduction including germination, root growth and the regulation of stomatal aperture. Taken together, our data suggest an important role for NPX1 as a negative regulator of ABA signaling.

Plants Overexpressing NPX1 Show Hypersensitivity to Drought Stress
The control of water loss from the leaf surface triggered by ABA is a crucial survival mechanism for plants during drought periods. To investigate the role of NPX1 in regulating drought tolerance and water loss, the soil of 3-week-old plants was covered with plastic and water was withheld for two weeks. NPX1-ox plants wilted more quickly than the other lines whereas npx1-1 remained turgid longer (Fig. 3A). Since survival depends on the rate at which water is depleted from pots, we also measured the stomatal conductance 3 d and 7 d after watering was stopped and calculated the ratio of stomatal conductance to soil moisture. This provides a measure of the sensitivity of stomatal closure to soil moisture or stomatal response to soil water deficit. Three days after watering was stopped, the soil moisture levels in the pots were still relatively high ( Fig. 3C) but the npx1-1 plants had significantly lower rates of water-loss (Fig. 3B). In contrast, the transpiration rate of NPX1-ox plants was not significantly different than the wild type (Fig. 3B). However, after 7 d NPX1-ox plants showed a 1.4-fold increase in water-loss compared to the wild type and npx1-1 (Fig. 3 2C). From these results, we conclude that overexpression of NPX1 reduces sensitivity to ABA and so those plants lose water faster and are more drought sensitive than the npx1-1 and wild type.

NPX1 Is a Nuclear Protein
We identified three putative nuclear localization sequences (NLS; softberry ProtComp6.0 http://www.softberry.com/berry.phtml?topic=protcompan&group=help& sub group =proloc) in the C-terminal region of NPX1 which suggested that NPX1 could be localized to the nucleus. Therefore, we tested the cellular localization of NPX1 fused to yellow also showed that the YFP signal appeared only in the nucleus.

NPX1 Regulate the Expression of ABA-Signaling Genes
Since results suggested that NPX1 may be involved in ABA signaling and was localized to the nucleus, we tested whether NPX1 is involved in regulating the expression of genes important for ABA-signaling in the wild type, NPX1-ox and npx1-1 before and after the treatment of 100 μ M ABA. Our results clearly show that the expression of AREB1, RD29A and ABI1 was down-regulated in the NPX1-ox without ABA treatment (Fig. 5, A and B). In contrast, the expression of ABA biosynthesis genes NCED3 and ABA2 as well as AtrbohF was up-regulated in the NPX1-ox without ABA treatment (Fig. 5C). In the wild type, the expression of MYB2, AtrbohD and OST1 is up-regulated after the treatment of ABA (Fig. 5). However, the induction of these genes was greatly attenuated or abolished in NPX1-ox. The AtrbohF gene was expressed at higher levels in the NPX1-ox lines even without the application of exogenous ABA and the expression pattern was opposite to AtrbohD (Fig. 5

NPX1 Contributes to the Regulation of ABA Bosynthesis
The fact that NPX1 is co-expressed with ABA biosynthesis gene NCED3 (Ma and Bohnert, 2008) and the expression of NCED3 and ABA2 is up-regulated in the NPX1-ox without ABA treatment ( Fig. 5C) suggests that NPX1 may be involved in the regulation of biosynthesis of ABA. To investigate this possibility, we measured ABA in wild type, NPX1-ox and npx1-1 using multiple plants in three sets of independent biological replicates. The ABA levels were significantly higher in NPX1-ox than other lines (Fig.   6A). We also measured ABA levels in leaves and roots after K + starvation. The level of ABA in leaves increased 6 h and 30 h after deprivation; whereas the production of ABA in roots increased only 30 h after deprivation ( Fig. 6B) which confirms previous results in Ricinus communis (Peuke et al., 2002) and helps to explain why this gene was upregulated under potassium deprived conditions.

NPX1 Interacts with TIP and Represses Transcription in Yeast
Because NPX1 was localized to the nucleus and the expression of several genes involved in ABA biosynthesis and signaling was altered in NPX1-ox plants, we tested the transcriptional activation of NPX1 even though it didn't contain a classical DNA binding domain (BD). For this experiment, NPX1 was fused with GAL4 BD and transformed with GAL4 activation domain (AD) containing empty vector. NPX1 did not show transcriptional activation activity in yeast (data not shown). Therefore, we tested whether NPX1 may regulate gene expression through interactions with other proteins such as transcription factors. To identify proteins that interact with NPX1, we used the yeast two-hybrid system with NPX1 fused in-frame to the GAL4 BD (BD-NPX1). First, we checked for interactions between BD-NPX1 and GAL4 AD fused to eight different transcription factors that were already known to be regulators of ABA signaling: AREB1, Nilson and Assmann, 2007). However, none of these eight candidates interacted with NPX1 (data not shown). Therefore, we screened an Arabidopsis root library fused to the GAL4 AD. Of the 1.5 X 10 6 transformants screened, 44 colonies initially grew on plates without Leu, Trp, and His. When these 44 clones were re-transformed and subjected to colony-lift filter assay, 20 clones exhibited β -galactosidase activity. The cDNAs were recovered from these 20 colonies and sequenced (Supplemental Table. S1). One of the cDNAs encoded a NAC transcriptional factor previously named TIP (Ren et al., 2000).
The interaction between NPX1 and TIP was confirmed using additional plasmid combinations and constructs to test for the following: auto-activation of the HIS3 and lacZ reporter genes, possible direct interactions with the GAL4 BD or AD, and potential artifacts caused by high expression of the encoded GAL4 fusion protein. BD-NPX1 alone did not activate the HIS3 or lacZ reporter gene whereas the co-transformation of BD-NPX1 and AD-TIP activates HIS3 or lacZ reporter gene (Fig. 7A). This interaction was confirmed by quantitative β -galactosidase assays using o-nitrophenyl-β-dgalactopyranoside (ONPG) as a substrate. The β -galactosidase activity of NPX1 and TIP co-transformed cells was about 34% of that observed for the positive control (BD-Krev1 and AD-RalGDS-wt, data not shown).
To reveal the underlying molecular mechanism of interaction between NPX1 and TIP, we tested whether this direct interaction could lead to activation or repression of the reporter gene expression in yeast. Yeast containing an integrated GAL4-lacZ reporter was transformed with BD-TIP in the presence or absence of BD-NPX1. BD-TIP was previously reported to activate a lacZ reporter (Ren et al., 2000), and we confirmed this activation (Fig. 7B). The activity of BD-TIP was signficantly reduced by 54 % when BD-NPX1 was present but not significantly reduced by BD-YFP which was used as a control ( Fig. 7B). Taken together, our results indicate that NPX1 interacts with TIP and functions as a transcriptional repressor.

NPX1 Is a Novel Protein involved in ABA responses
The previously unknown gene NPX1 was identified as a gene whose expression was upregulated by potassium deficiency (Shin and Schachtman, 2004 expression was lower when ABA was added (Fig. 5). We also showed that the overexpression of NPX1 inhibits the expression of ABA-induced OST1 and AREB1 (Fig.   5) which suggests that the decreased stomatal sensitivity to ABA in NPX1-ox might be caused in part by the repression of drought stress-regulated genes such as OST1 and AREB1. Expression of AtbohD is also regulated by NPX1, but there appears to be some compensation in the form of the increased expression of AtbrohF. We have seen this type of compensation in the expression of NADPH oxidase family members in roots of the rhd2 mutant (Shin et al., 2005). AtERF7 targets PKS3 which are Ser/Thr protein kinases in the pathway (Song et al., 2005). This is the first report, to our knowledge, of an upstream transcriptional regulator of the OST1-mediated signaling pathway.
ABA also induces expression of stress-related genes (Shinozaki and Yamaguchi-Shinozaki, 2007) such as RD29A. More than half of drought-inducible genes such as RD29A are also induced by high salinity and/or ABA treatment, implicating significant cross-talk between the drought, high salinity (Shinozaki and Yamaguchi-Shinozaki, 2007) and ABA response pathways. In our study, the ABA-inducible NPX1 was also upregulated by NaCl and cold stress (Fig. 1), and NPX1-ox plants were hypersensitive to drought stress (Fig. 3). The regulation of RD29A is enhanced in npx1-1 plants without the addition of ABA, but upon addition of ABA RD29A expression increases in all lines tested. This suggests that either the application of exogenous ABA short circuits NPX1 effects or that the effects of NPX1 are subtle and tightly regulated at endogenous levels of ABA.
The regulation of gene expression under drought stress is mediated by multiple transcriptional cascades (Zhu, 2002;Yamaguchi-Shinozaki and Shinozaki, 2006). In each cascade, a set of transcription factors are induced by ABA, which in turn activates or represses downstream target genes important for drought resistance (Shinozaki and Yamaguchi-Shinozaki, 2007). From our results we conclude that NPX1 negatively regulates ABA signaling in part by reducing the expression of ABA-inducible transcription factors such as AREB1 and MYB2 and stomata closure by regulating ABI1 and OST1 (Fig. 5). Thus, NPX1-ox plants are hypersensitive to drought whereas npx1-1 plants are more tolerant (Fig. 3). ABI1 is known as a negative regulator in stomatal closing and has been shown to be a negative regulator of OST1 (Yoshida et al., 2006).
The changes in expression of MYC2 with added ABA and the lower expression in the  . 5C and 6A). Even though levels of ABA are higher in the NPX1-ox, these plants appear to be relatively less sensitive to ABA. Therefore we suggest that NPX1 does not merely contribute to ABA signaling by increasing ABA content. Rather we propose that the increased ABA concentrations are due to feedback which may be initially caused by insensitivity which triggers increased ABA biosynthesis to overcome a perceived reduction in sensitivity. The idea of this type of feedback was suggested earlier (Xiong et al., 2001) and our study provides evidence that this occurs. The observation that NPX1-ox is less sensitivity to, but contains more ABA, differs from other mutants such as ahg2 which accumulates 50% higher endogenous ABA but appears to be more hypersensitive to ABA (Nishimura et al., 2005).
In contrast the sad1 mutant shows enhanced sensitivity to ABA but does not produce more ABA under stress conditions as compared to the wild type (Xiong et al., 2001). In the case of sad1 the authors suggested that ABA production was reduced in sad1 which resulted in enhanced sensitivity to ABA and abiotic stress. Multiple results from different groups suggest that ABA responses are complex and regulated at different levels including direct effects and feedback responses. Our results suggest that NPX1 modulates ABA signaling through effects on the signal transduction pathway, but the possibility still exists that it could act indirectly by inducing ABA biosynthesis. Thus, we conclude that NPX1 reduces ABA sensitivity through the repression of stress-inducible gene expression but activates the induction of ABA biosynthesis via positive feedback regulation.

A NAC Transcriptional Factor Is a Target of NPX1
To identify how NPX1 modulates ABA signaling and since our hypothesis was that modulation occurred through protein/protein interactions rather than through direct binding to DNA we employed yeast two-hybrid assays. We found that NPX1 Fig. S4).
Furthermore, we identified regions in the promoters of OST1 and AREB1 that contained a CATGTG motif (data not shown) which have been shown to be NAC binding motifs (Tran et al., 2004). Since TIP activates reporter gene expression in yeast cells (Ren et al., 2000) we used a yeast repressor assay to test whether NPX1 may directly repress TIP activity. We showed that TIP activity was reduced when NPX1 was expressed in the same yeast cells (Fig. 7B). The ABI1 expression was reduced in the tip plants without added ABA and OST1 expression was lower when ABA was added (Supplemental Fig.   S5B). The regulation of these two genes in the tip knock-out is as we would predict based on our results in NPX1-ox plants. (Fig. 5).  Fig. S3). It is well known that root growth is stimulated by ABA when applied at a low concentration, but it inhibits growth at higher concentrations (Pilet and Saugy, 1987). It is possible that since ABA is needed for growth under normal conditions and that higher concentration may be required for maintaining root growth under potassium-deprived conditions. This finding links ABA to the potassium deprivation response circuits which include auxin (Shin et al., 2007), ethylene (Jung et al., 2009) and ROS (Shin and Schachtman, 2004). Further work will be needed to fully understand the role of ABA plant response to low potassium.
In conclusion, the data presented here provide evidence that a novel nuclear factor modulates ABA signaling and plant sensitivity to abiotic stress as summarized in Fig. 8.
The isolation and characterization of NPX1 provides new insight into the control mechanisms for modulating the ABA responsiveness of plants and may provide new approaches for the genetic engineering of drought tolerance in plants.

DNA cloning
The NPX1 (At5g63320) open reading frame was amplified using Pfu-polymerase

Quantitative real-time PCR analysis
To analyze the expression of NPX1 by real-time PCR, the gene-specific primers (  and was used to normalize the reactions. Real-time PCR was performed according to the instructions provided for the Bio-Rad iCycler iQ system (Bio-Rad laboratories) with platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The fold change of transcripts was calculated based on an efficiency calibrated model (Yuan et al., 2006) and compared with the transcript level under normal condition. Statistical differences between samples were evaluated by Student's t-test using delta Ct values (Yuan et al., 2006). In each experiment, the mean of three biological replicates are used to generate means and statistical significance. Two experiments on independently grown plant material were performed to confirm that the results are reproducible.

Plant material and growth conditions
For constitutive expression of NPX1 in planta, we used pCambia2300 with the figwort mosaic virus (FMV) promoter (Sanger et al., 1990) and nopaline synthase terminator.
Plants were transformed using Agrobacterium tumefaciens by the floral dip method (Clough and Bent, 1998

Germination and root growth assays
Germination and root growth assays with exogenous ABA were performed as described (Kuhn et al., 2006), Sterilized seeds were plated on minimal medium (0.25× Murashige and Skoog medium, no Suc) supplemented with increasing ABA concentrations. To score seed germination, the percentage of seeds that had germinated and developed fully green expanded cotyledons were determined in three independent experiments (36 seeds per genotype and experiment).
Root growth assays to assess ABA sensitivity were carried out by transferring 6d-old seedlings onto minimal medium supplemented with the indicated ABA concentrations on 0.8% agar (Phytagel; Sigma) plates. Root growth was measured 6 d after the transfer in three independent experiments with seventy individuals per genotype and experiment.

Measurements of the stomatal aperture
For stomatal closing experiments, fully expanded leaves from 3-to-4-week-old plant were excised, and epidermal pieces were peeled from the abaxial surface. The epidermal peels were floated for 2.5 h in stomatal opening solution (Pei et al., 1997) containing 50 mM KCl, 50 μ m CaCl 2 , and 10 mM MES (pH 6.15). After incubation in ABA for 2 h, the stomatal aperture was measured. Control experiments were performed in parallel with no ABA added. Double-blind stomatal movement assays were performed such that the genotype and applied ABA concentrations were unknown. Three independent experiments were performed, and the same results were obtained.

Drought tolerance experiment
For the drought tolerance experiment, the soil in pots containing 3-week-old plants were covered with plastic film and were exposed to a period of 14 d without added water.
Stomatal conductances (gs) were obtained using AP3 porometer (Delta-T Devices, Burwell, Cambridge, UK) after 3 d and 7 d after watering was stopped. Two independent experiments were performed, and similar results were obtained.

Visualization of YFP-NPX1 localization
For transient expression of YFP-NPX1, biolistic bombardment of onion epidermal cells was performed as previously described (Marc et al., 1998)

Yeast repression assay
Full-length TIP (At5g24590) cDNA was generated by PCR with the following primers AGAATTCATGAAAGAAGACATGGAAGTACT and ACTCGAGGAATTGATCG GAACAAACATCAC and subcloned into the EcoRI/SalI sites in pGBKT7 (Clontech). As a result, TIP was fused with GAL4 DNA-binding domain (BD). BD-NPX1 and BD-YFP were expressed from pDEST22 (Invitrogen). The plasmids were transformed into S.
cerevisiae Y187 and ß-galactosidase assays were performed as described (Sridhar et al., 2006). BD-TIP, BD-NPX1 or BD-YFP was selected by -Trp and -Leu. A liquid culture assay using o-nitrophenyl β -d-galactopyranoside as a substrate was performed to determine transcriptional activation. Data shown for these assays are averages of triplicates, and the experiment was repeated twice.

Supplemental Data
Supplemental Figure S1. RT-PCR analysis of NPX1 expression in Arabidopsis plants.
Tissues analyzed were roots, rosette leaf, cauline leaf, stem, flower and developing Supplemental Table S1. Proteins that interact with NPX1 as determined by yeast two hybrid screening.
Supplemental Table S2. Information on Genes used for real-time PCR analysis.     the soil moisture during drought stress treatment (n = 20. for each group). "a" indicates the values are presented as mean ± SEM. and NYFP-NPX1 (bottom). Signals from bright-field (bright) and NYFP (YFP) are shown.

Figure Legends
B and C, NPX1 localization in stable transgenic Arabidopsis leaves (B) and roots (C).
Signals from bright-field (bright) and NYFP (YFP; green), and Hosecht signal (red) indicate nuclear localization and the merge of the three signals (merge; yellow) are shown. or ABA biosynthetic pathway (C). Different letters indicates a significant difference between means at P < 0.05 (Student's t-test). See Table S2 for full information on the genes represented in this figure.  respectively. White colonies indicate a lack of reporter activation. B, NPX1 represses the activity of TIP. BD-NPX1, represses the activity of TIP in yeast, as indicated by the decreased ß-galactosidase activity. BD-TIP, but not NC (co-transformants of pGBKT7 and pDEST 32 vector) and BD-NPX1, activate lacZ expression. Data represent the mean ± standard deviation of three independent experiments. Different letters indicates a significant difference between means at P <0.05 (Student's t-test).