Plant Mol Biol (2011) 75:451–466 DOI 10.1007/s11103-011-9748-2
Arabidopsis SAP5 functions as a positive regulator of stress responses and exhibits E3 ubiquitin ligase activity Miyoung Kang • Mohamed Fokar • Haggag Abdelmageed • Randy D. Allen
Received: 17 September 2010 / Accepted: 15 January 2011 / Published online: 4 February 2011 Ó Springer Science+Business Media B.V. 2011
Abstract AtSAP5, one of approximately 14 members of the Stress Associated Protein gene family in Arabidopsis, was identified by its expression in response to salinity, osmotic, drought and cold stress. AtSAP5 shows strong homology to OSISAP1, an A20/AN1-type zinc finger protein implicated in stress tolerance in rice. To evaluate the function of AtSAP5 in the regulation of abiotic stress responses, transgenic Arabidopsis plants that over-express AtSAP5 (35S::AtSAP5) were characterized, along with wild-type and T-DNA knock-down plants. Plants that overexpress AtSAP5 showed increased tolerance to environmental challenges including salt stress, osmotic stress and water deficit. Comparison of gene expression patterns between 35S::AtSAP5 transgenic plants and wild-type plants under normal conditions and water deficit stress indicated that over-expression of AtSAP5 correlates with up-regulation of drought stress responsive gene expression. Analysis of transgenic plants that express GFP-AtSAP5 showed that it is localized primarily in nuclei of root cells and recombinant AtSAP5 has E3 ubiquitin ligase activity
Electronic supplementary material The online version of this article (doi:10.1007/s11103-011-9748-2) contains supplementary material, which is available to authorized users. M. Kang H. Abdelmageed R. D. Allen (&) Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA e-mail:
[email protected] M. Kang H. Abdelmageed R. D. Allen Institute for Agricultural Biosciences, Oklahoma State University, Ardmore, OK 79413, USA M. Fokar Center for Biotechnology and Genomics, Texas Tech University, Lubbock, TX 79409, USA
in vitro. These results indicate that AtSAP5 has E3 ligase activity and acts as a positive regulator of stress responses in Arabidopsis. Keywords AtSAP5 A20/AN1 zinc finger protein Abiotic stress E3 ubiquitin ligase Abbreviations SAP Stress Associated Protein ABA Abscisic Acid RING Really Interesting New Gene TNF Tumor Necrosis Factor
Introduction A family of Stress Associated protein (SAP) genes, some of which appear to play important roles in the regulation of plant responses to abiotic stress, was identified in Oryza sativa (Mukhopadhyay et al. 2004). The SAP gene family is conserved in angiosperms with 14 SAP genes identified in the Arabidopsis genome, 11 in Zea mays and 19 in Populus trichocarpa (Jin et al. 2007; Vij and Tyagi 2008). The most completely characterized SAP genes are OsiSAP1, OsiSAP8 and ZFP177 from rice. Although expression of these genes in rice is up-regulated by stress treatments that include salt, drought, cold, and ABA, the function of this gene family in other plant species is not clear (Huang et al. 2008; Kanneganti and Gupta 2008; Mukhopadhyay et al. 2004). The proteins encoded by most members of this gene family have two putative zinc finger motifs, an A20 zinc finger domain [Cys-X(2-4)-Cys-X11Cys-X2-Cys] at the N-terminus and an AN1 zinc finger
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domain [Cys-X2-Cys-X(9-12)-Cys-X(1-2)-Cys-X4-CysX2-His-X5-His-X-Cys] at the C-terminus (where X can be any amino acid). Genes with a similar combination of zinc finger domains are also present in mammals. While the A20/AN1 zinc finger proteins are known to regulate NF-jB activation in mammalian cells through ubiquitin-dependent mechanisms, the regulatory targets and modes of action of plant SAP proteins are unknown. HsA20, a protein that contains seven A20 zinc finger motifs, was discovered in human endothelial cells. Expression of HsA20 is induced by tumor necrosis factor (TNFa) (Dixit et al. 1990; Opipari et al. 1990); although initially thought to be a transcription factor, subsequent research showed that A20 ubiquitinates RIP and inhibits NF-jB activation (Heyninck et al. 1999; Song et al. 1996). Subsequently, A20 protein was shown to have dual ubiquitin editing functions required for the down-regulation of NF-jB signaling. In addition to E3 ubiquitin ligase activity that is mediated through the fourth A20 zinc finger domain, the A20 protein contains an OTU (ovarian tumor) domain that is involved in de-ubiquitination (Wertz et al. 2004). Other well studied A20-like proteins in mammalian cells include ZNF216 and Rabex-5. Unlike A20, these proteins have a single A20 zinc finger domain, along with a second zinc finger motif, either AN1 or IUIM, respectively (Huang et al. 2004; Mattera et al. 2006). Although ZNF216 has both A20 and AN1 zinc finger domains, Hishiya et al. (2006) reported that it does not have detectable E3 ubiquitin ligase activity. However, ZNF216 can bind directly to polyubiquitin chains via its A20 zinc finger domain and deliver polyubiquitinated substrate to the 26S proteasome (Hishiya et al. 2006). Although the exact molecular function of the AN1 zinc finger domain of ZNF216 is not known, it was found to be involved in the inhibition of NF-jB activation through interaction with TRAF6 (Huang et al. 2004). Rab5 guanine nucleotide exchange factor (Rabex-5) interacts with ubiquitin through its A20 zinc finger domain and a novel inverted ubiquitin interacting motif (IUIM) at its N terminus. Rabex-5 has E3 ligase activity and recruits ubiquitin-loaded E2 conjugating enzyme through its A20 zinc finger motif (Lee et al. 2006; Mattera et al. 2006; Penengo et al. 2006). Ubiquitination, a critical regulatory mechanism in all eukaryotic cells, is a posttranslational modification system that is involved in protein quality control and protein stability through the covalent attachment of a 76-amino acid ubiquitin polypeptide to a target protein (Ben-Neriah 2002). This pathway requires three enzyme activities, ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2), and ubiquitin ligases (E3). Single unit E3 ubiquitin ligases are divided into two groups, which are defined by the presence of either a HECT or a RING finger domain. RING finger E3 ubiquitin ligases interact with a
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substrate protein and an E2 enzyme, while HECT domain E3 ubiquitin ligases form covalent thiolesters with ubiquitin and directly transfer it to Lys residues of the substrate protein. In Arabidopsis thaliana, more than 1,400 genes encode components of ubiquitin/proteasome pathway and 90% of these genes encode subunits of E3 ubiquitin ligases (Moon et al. 2004; Smalle and Vierstra 2004). The wide diversity of E3 ubiquitin ligases suggests that they have evolved distinct substrate specificities (Vierstra 2003). In plants, ubiquitin-mediated protein modifications have been shown to play significant roles in multiple cellular functions including photomorphogenesis (Yang et al. 2005), pathogen defense (Yang et al. 2006; Zeng et al. 2004), hormone responses (Xie et al. 2002) and regulation of abiotic stress acclimation (Cho et al. 2008; Dong et al. 2006; Qin et al. 2008; Yan et al. 2003; Zhang et al. 2007). We report here that AtSAP5 encodes a protein with both A20 and AN1 zinc finger motifs with E3 ubiquitin ligase activity. Expression of AtSAP5 is induced by various stresses in Arabidopsis and elevated expression of AtSAP5 in transgenic plants results in altered gene expression patterns and leads to increased tolerance to water deficit stress. Thus, AtSAP5 appears to plays an important role in the acclimation of plants under stressful conditions.
Materials and methods Plant materials and growth conditions Wild-type, Arabidopsis thaliana (ecotype Columbia), 35S:AtSAP5 transgenic plants and T-DNA insertion mutant lines were used for this study. Seeds of the T-DNA insertion lines (Salk_073787 and Salk_073784) were provided by the Arabidopsis Biological Resource Center (ABRC). Seeds were sterilized and grown on soil or 0.59 MS media containing 0.8% agar. The plates were stratified for 3 days at 4°C and then transferred into a growth chamber at 20–22°C with a 16-h photoperiod. For abiotic stress treatments, 2-week-old seedlings were exposed to dehydration by transferring seedling to filter paper in a covered Petri dish, 4°C (low temperature treatment), or 600 lmol m-2 s-1 (high light stress). For ABA, NaCl, mannitol and H2O2 treatments, the plants were submerged in solutions containing 100 lM ABA, 250 mM NaCl, or 300 mM mannitol, or 10 mM H2O2. After the treatment, seedlings were sampled at the indicated time for RNA analysis. Over-expression of 35S::AtSAP5 A full-length AtSAP5 cDNA was amplified from stock DNA obtained from ABRC (At3g12630) using the
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following primers: forward primer, 50 -CCCTCTAGAGAA TTCATGGCTCAGAGAACGGAGAAGG-30 and reverse primer, 50 -CCCGGATCCTTAAACTTTGACCATTTTC GC-30 , and PCR products were ligated into the pGEM-T Easy vector (Promega). The vector was digested with XbaI and BamHI and inserted into the pBI121 binary vector linearized by a double digestion using the same restriction enzymes. Transformation of Arabidopsis was performed according to the flower -dip method using Agrobacterium tumefaciens GV3101. Transformants were selected on 0.59 MS medium containing kanamycin (50 mg/l).
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with A. tumefaciens strain GV310. To detect b-glucuronidase (GUS) activity, the transgenic plants were placed in GUS staining solution (1 mM 5-brom-4-chloro-3-indolylb-D-glucuronic acid, 0.1 M Na2H2PO4, 10 mM Na2 EDTA, 0.5 mM K ferrocyanide, 0.5 mM K ferricyanide, 0.1% Triton X-100, pH7.0) and vacuum infiltrated to remove trapped air for 20–30 min. The tissues were then incubated at 37°C overnight. Chlorophyll was removed by washing with 70% ethanol. Plants were visualized with a dissecting microscope (Olympus SZX 12). Subcellular localization of GFP-AtSAP5
Stress tolerance tests The sensitivity of seed germination and seedling growth to osmotic stress was tested on 0.59 MS medium containing 350 mM mannitol or 125 mM NaCl. To test germination, about thirty seeds were sown on 0.59 MS medium containing osmotic chemicals and then, the number of seeds germinated was scored as rate of germination (percent seeds germinated compared to seeds from the same lot with no stress treatment). For root elongation measurements, seeds were grown on plates with 0.59 MS medium placed vertically for 4 days and then transferred to 0.59 MS medium containing mannitol or NaCl. Root length was measured after 6 days of growth. For drought tolerance analysis, plants were grown in soil for 3 weeks at 22°C. Water was withheld for 2 weeks, and then the plants were rehydrated. To measure the water loss, detached fresh leaves were placed abaxial side up on weighing dishes and allowed to dry at room temperature. Leaf weight was measured every 30 min for 2 h, and then every 1h thereafter. To measure stomatal closure, leaves of the same developmental stages were kept under dark condition for 3 h, and incubated under light for 1 h. Leaf peels were prepared and stomatal apertures were observed using a Microphot-FX microscope (Nikon) coupled to a CCD camera. Histochemical GUS assay The 2 kb promoter fragment (-99 to -2099 position from ATG upstream) including the 50 flanking region of the AtSAP5 gene was amplified from genomic DNA using the following primers: forward primer, 50 -CGGGATCCC GAACATAGATAGTCCCAAACACACAC-30 and reverse primer, 50 -CCCCCGGGGGATCATAGAGAGACGCGTT TCCATCTC-30 . The amplified promoter region was ligated into the pGEM T-easy vector and excised with BamHI and SmaI, and then subcloned into pBI101 vector. The accuracy of the insert was verified by DNA sequencing. This construct was introduced into wild-type Arabidopsis plants
For construction of the GFP-AtSAP5 gene fusion, the AtSAP5 cDNA in pGEM T-easy vector plasmids were digested with BamHI and EcoRI and ligated into pEGAD vector. Insertion of AtSAP5 cDNA was confirmed by DNA sequencing. This construct was used to transform wild-type Arabidopsis plants using A. tumefaciens strain GV310. T2 transgenic lines resistant to Basta were selected and analyzed. For visualization, roots of 5-day-old seedlings were stained with propidium idodide (1 lg/ml). GFP fluorescence of transgenic plants was observed using a laser scanning confocal microscope (Leica DM IRE2). Microarray and real-time PCR analysis Wild-type and 35S::SAP5 plants for microarray experiment were grown in a growth chamber (22°C, 16 h light/8 h dark). Two week-old seedlings were harvested and RNA was extracted using the Total RNA Isolation Kit (Qiagen). Ten micrograms of total RNA from each sample was analyzed using the ATH1 Affymetrix array. Probe labeling and hybridization were carried out at the Noble Foundation Microarray Facility according to manufacturer’s procedures. For quantitative real-time RT PCR, DNase I-treated total RNA (1 lg) was used to synthesize cDNA using oligo (dT) and Iscript reverse transcriptase (Bio-Rad). Real-time PCR amplification experiments and calculations of relative expression levels were performed following User Bulletin # 2 ABI PRISM7000 Sequence Detection System (Applied Biosystems) with iTag SYBR Green Supermix (Bio-rad). PrimerQuest (Integrated DNA Technologies) was used to design primers for Real-time PCR. Arabidopsis ubiquitin 9 (UBQ 9) was used as normalizer in each experiment. Relative transcript abundance was calculated using the Ct method. After calculation of DCt (Ct; expression of interest gene—Ct; expression of UBQ9), the dCt value of the calibrator sample (the sample with the highest dCt value) was subtracted from every other sample to produce the ddCt value. In most of our experiments, control sample had highest d Ct value; thus, we used it as the calibrator sample.
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2-DDCt was used for every sample as the relative expression level. Duplicate biological replications and triplicate technical replications were performed for each data point. Data are presented as expression ratios between genotypes and stress treatments. Primers used for qRT-PCR are shown in Supplemental Table 1 in Online Resource 1. Recombinant protein expression and purification Full length AtSAP5 cDNA or truncated cDNA was recombined into the pDEST15 vector via the Gateway system (Invitrogen) to produce the N-terminal GST fusion protein. The plasmids were expressed in BL21-AI cells. E. coli clones were grown in LB medium containing 50 lg/ml carbenicillin to a density of OD600 = 0.6. Expression of recombinant protein was induced for 6 h at 28°C with 0.2% L-arabinose and 0.1% glucose. Protein was extracted by using CelLytic B cell Lysis buffer (Sigma-Aldich). Recombinant fusion proteins were purified by GST-binding kit (Novagen). E3 ubiquitin ligase activity assay In vitro ubiquitination assays were conducted as described previously (Wertz et al. 2004). Each reaction (25 ll final volume) contained 20 mM HEPES, pH 7.2, 100 mM NaCl, 5 mM MgCl2, 5 mM ATP, 10 mM DTT, 5 lg HA-ubiquitin (Boston Biochem), 150 nM yeast E1 (Boston Biochem), 200 nM human recombinant UbcH2 (Boston Biochem), and purified 5 lg GST-AtSAP5. The reaction mixture was incubated at 30°C for 2 h and stopped by adding 59 SDS–PAGE sample buffer (125 mM Tris–HCl, pH 6.8, 20% glycerol, 4% SDS, and 10% b-mercaptoethanol) and boiled at 100°C for 5 min. Protein samples were analyzed by SDS–PAGE electrophoresis followed by protein gel blotting. Blots were probed using anti-HA antibodies (Sigma) or anti-GST antibodies (Invitrogen), followed with anti-mouse AP conjugate (Invitrogen).
Results Expression of AtSAP5 AtSAP5 encodes a member of the stress associated protein (SAP) family (Vij and Tyagi 2006) that is expressed throughout Arabidopsis plants (Fig. 1a). Analysis of Genenvestigator data indicated that expression of AtSAP5 is strongly induced by a variety of stress treatments. To verify whether the expression of AtSAP5 is regulated by abiotic stresses, 2 week old Arabidopsis seedlings were subjected to various treatments and RNA was prepared for quantitative real-time PCR analysis (Fig. 1b). Expression
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of AtSAP5 was induced by more than fourfold in response to cold or ABA treatments, twofold by NaCl stress and ninefold by mannitol treatment. In silico analysis of the *2 kb promoter region using the PLACE promoter analysis program (Higo et al. 1999) detected several cis-acting elements, including an ABA response element (ABRE), drought response element (DRE), low temperature response elements (LTRE), and MYB/MYC elements, which are known to mediate transcriptional responses to different stress signals. To investigate the sensitivity of the AtSAP5 promoter to abiotic stress, T2 generation transgenic plants that express a reporter gene consisting of the AtSAP5 promoter and approximately 2 kb of 50 flanking sequence fused to the b-glucuronidase (GUS) coding sequence (AtSAP5p::GUS) were examined. GUS expression was strongly induced by 300 mM mannitol treatment in seedlings of several AtSAP5p::GUS transgenic lines (Fig. 1c). Quantitative analysis of the changes in GUS mRNA in these plants in response to various stress treatments indicated stress-responsive up-regulation of the reporter gene (Fig. 1d). These results confirm that the induction of AtSAP5 in response to abiotic stress is transcriptionally regulated.
35S::AtSAP5 transgenic plants show osmotic stress tolerance A reverse genetic approach was used to investigate the biological function of AtSAP5. Transgenic Arabidopsis plants were generated that constitutively expressed AtSAP5 under control of the cauliflower mosaic virus 35S promoter (35S::AtSAP5). In addition, two independent T-DNA insertion lines, atsap5-1 (Salk_073787) and atsap5-2 (Salk_073784) were obtained from the ABRC seed stock center. The T-DNA insert positions in these lines are located within the promoter region of the AtSAP5 gene (see Supplemental Fig 1 in Online Resource 1). Expression of AtSAP5 in transgenic 35S::AtSAP5 and T-DNA mutant plants was verified by qRT-PCR analysis (Fig. 2a). Two individual 35S::AtSAP5 transgenic lines (35S::AtSAP5-a and 35S::AtSAP5-b) that had AtSAP5 transcript levels more than 10-fold higher than wild-type plants were selected for further investigation (Fig. 2a). In T-DNA mutant lines, expression of AtSAP5 was reduced by approximately 30% in atsap5-1 plants and by 60% in atsap5-2 plants, relative to wild-type plants, indicating that these T-DNA insertions result in knockdown, rather than knockout phenotypes (Fig. 2a). Compared to wild-type plants, no morphological or developmental alterations were seen in 35S::AtSAP5 transgenic or atsap5 knockdown plants under normal growth conditions (see Supplemental Fig 2 in Online Resource 1).
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Fig. 1 Expression pattern of the AtSAP5. a RT-PCR analysis of AtSAP5 expression in a whole Arabidopsis plants and in a variety of organs, as indicated. b Quantitative RT-PCR was conducted to determine the level of AtSAP5 transcripts under a range of stress treatments. Control, (no treatment), Salt (250 mM NaCl); Heat (42°C); HP (10 mM H2O2); high light (HL, 600 lmol m-2 s-1); ABA (100 lM); Chilling (4°C for 12 h); Mannitol (300 mM). Bars
indicate SD. Mean vaules and standard errors were obstained from three technical replications. c Histochemical GUS staining of transgenic Arabidopsis seedlings containing the AtSAP5 promoter::GUS fusion gene. Plants were grown with no treatment (Control) or with 300 mM mannitol, as indicated. d Quantitative RT-PCR analysis of GUS mRNA expression in AtSAP5p::GUS Arabidopsis plants in response to stress treatments (as above)
Fig. 2 Osmotic and salt stress tolerance of 35S::AtSAP5 and T-DNA mutant plants. a Transcripts of AtSAP5 in wild-type, 35S::AtSAP5 and atsap5 mutant plants by qRT-PCR. Bars indicate SD from three technical replications. b After independent 35S::AtSAP5 plants (a and b) and mutant plants (atsap5-1 and atsap5-2), and wild-type plant were incubated for 3 days at 4°C, seeds were placed at 22°C and the percentage of germinated seeds were determined after 3 days on 0.5X MS media containing either 350 mM mannitol, or 150 mM NaCl. Bars indicate SD from three technical replications. c Root elongation
of wild-type, 35S::AtSAP5 transgenic plants, and knockdown mutant plants after 6 days growth on 0.5X MS agar plate with mannitol or NaCl (as above). Bars indicate SD, **P \ 0.05 (Tukey HSD multiple group test was used). d Effect of osmotic stress on post-germinative development. Data were collected 10 days after germination. Values are means of percentage of cotyledon numbers showing greening (triplicate, n = 30 each). Bars indicate SD, P \ 0.05 (Tukey HSD multiple group test)
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To investigate the role of AtSAP5 in determining the sensitivity of Arabidopsis plants to osmotic stress, seeds of 35S::AtSAP5, atsap5 knockdown and wild-type plants were sown on MS agar media containing either 350 mM mannitol or 125 mM NaCl. The rate of seed germination (percent seeds germinated compared to control), extent of root elongation and cotyledon greening were observed and recorded. Under the conditions used, 70% of wild-type seeds germinated on mannitol-containing media and 78% germinated on media with NaCl. Seeds of the two individual 35S::AtSAP5 lines (a and b) showed 81 and 97% germination on media containing mannitol and 81 and 91% germination on media with NaCl. Only 63% of atsap5-1 and 36% of atsap5-2 seeds germinated on mannitol and 51% of atsap5-1 and 13% of atsap5-2 seeds germinated on NaCl (Fig. 2b). Thus, elevated expression of AtSAP5 enhanced seed germination under osmotic or salt stress while seeds with reduced AtSAP5 expression were more sensitive to these conditions. To determine the effects of AtSAP5 expression on seedling development, 4-day-old Arabidopsis seedlings were transferred onto media containing 350 mM mannitol or 125 mM NaCl and the length of the primary root and development of chlorophyll in cotyledons were monitored after 6 days of growth. Root growth and cotyledon greening of 35S::AtSAP5 plants were less sensitive to osmotic stress compared to wild-type and atsap5 plants (Fig. 2c, d), while the response of atsap5 seedlings did not differ significantly from wild-type seedlings in root growth. Taken together, these results indicate that AtSAP5 expression is involved in controlling osmotic stress tolerance in Arabidopsis seedlings. Over-expression of AtSAP5 enhances tolerance to water deficit stress Because osmotic stress is often associated with drought, we examined the accumulation of endogenous AtSAP5 transcripts in Arabidopsis plants during dehydration. Total RNA used for this qRT-PCR analysis was isolated from wild-type Arabidopsis at intervals during dehydration treatment. As shown in Fig. 3a, expression of AtSAP5 was induced by as much as threefold after 4 h of dehydration treatment. To investigate whether altered expression of AtSAP5 affected tolerance to dehydration stress, plants with various levels of AtSAP5 expression were tested for their response to water deficit. Water was withheld from 3-week-old wild-type, 35S::AtSAP5-a, 35S::AtSAP5-b, and atsap5-2 plants grown in soil to introduce dehydration stress and the plants were then rehydrated. After 2 weeks without added water, wild-type and atsap5-2 mutants suffered severe wilting and desiccation damage while 35S::AtSAP5-a and 35S::AtSAP5-b transgenic
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plants showed delayed wilting and reduced desiccation damage (Fig. 3b). Water loss from wild-type, 35S::AtSAP5 transgenic and atsap5 plants was also determined by measuring changes in fresh weight of detached leaves (Fig. 3c). After 6 h of dehydration, 35S::AtSAP5-a and 35S::AtSAP5b plants retained 60 and 50% of initial fresh weight, respectively, while the fresh weights of wild-type and atsap5-2 mutant leaves were reduced to approximately 40% (Fig. 3c). These results provide evidence that over-expression of AtSAP5 confers increased tolerance to dehydration stress by reducing transpiration, relative to wild-type plants. Stomata were observed microscopically using epidermal peels from mature leaves of 35S::AtSAP5, wild-type and atsap5 plants and relative stomatal opening was measured (Fig. 3d). The results showed that leaves of 35S::AtSAP5 plants had reduced average stomatal apertures compared to wild-type and atasp5 plants. Over-expression of AtSAP5 alters expression of stress responsive genes To gain a better understanding of the role of AtSAP5 in stress response pathways, changes in genome-wide transcriptional profiles associated with AtSAP5 over-expression were studied. Microarray experiments were performed using Affymetrix ATH1 and probes derived from RNA samples from wild-type and 35S::AtSAP5 transgenic plants. Under control conditions, 60 genes were identified that were expressed at levels at least twofold higher in 35S::AtSAP5 transgenic plants than in wild-type plants (see Supplemental Table 2 in Online Resource 1), while 30 genes were expressed a lower levels (see Supplemental Table 3 in Online Resource 1). Interestingly, among the 90 genes differentially regulated in AtSAP5 over-expressing plants, 40 encode proteins previously reported to be involved in abiotic stress responsive pathways (Catala et al. 2007; Seki et al. 2001). For example, increased expression of 6 genes encoding UDP-glucosyl transferases (UGT), 3 glutathione-S transferase genes, and 4 dehydrogenase/reductase genes was seen in 35S::AtSAP5 transgenic plants (Table 1). In addition, expression of genes involved in anthocyanin biosynthesis, including MYB75/PAP1, MYB90/PAP2, and DFR, was significantly induced in 35S::AtSAP5 plants relative to wild-type plants. Also, drought and salt inducible genes, including AtGolS2 and AtCP1 were upregulated in these plants. These results indicate that AtSAP5 may be involved in the regulation of stress responsive genes under normal growth conditions. Microarray results for selected genes were verified using qRT-PCR (Fig. 4). Total RNA used for qRT-PCR analysis was isolated from 35S::AtSAP5 transgenic and wildtype plants grown with or without a 4 h dehydration stress treatment. The results presented in Fig. 4 are ratios
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Fig. 3 Over-expression of AtSAP5 enhances dehydration tolerance. a Expression of AtSAP5 under dehydration stress at indicated time. Bars indicate SD from three technical replications. b Dehydration tolerance test. Three-week- old wild-type, 35S::AtSAP5 over-expressing plants (a and b), and atsap5-2 knockdown mutant plants were subjected to drought stress for 2 weeks, followed by re-watering. c Transpiration rates. Water loss was monitored by measurement of fresh weight loss from detached leaves at same development age at
time intervals indicated. Bars indicate SD. Experiments were performed in triplicate, n = 6, each. **P \ 0.01 by t-test. d Stomatal guard cells in epidermal peels from leaves of wild-type (left), 35S:AtSAP5-a (middle), and atsap5-2 mutant plants (right) were observed by light microscopy. The graph in the lower panel shows mean size of stomatal apertures from epidermal peels. The mean ratio of width to length of at least 20 stomatal apertures was measured. Bars indicate SD
between expression levels in different genotypes and/or treatments. The first bar in each graph shows the ratio between 35S::AtSAP5 over-expressing plants and wildtype plants under control conditions (OE-C/WT-C), which reveals the constitutive effect of SAP5 over-expression on the expression of these genes. Since many of these genes were selected from microarray experiments, most are expressed in the transgenic lines at levels that are between twofold (SDR, UGT75B1) and 20-fold (AtGolS2) higher than in wild-type plants. Exceptions include Xero2 and MYB29, which are expressed at the same level or reduced levels in 35S::AtSAP5 plants relative to wild-type plants (MYB29 was selected for qRT-PCR assays because the microarray analysis showed it to be down-regulated in 35S::AtSAP5 plants). The second bars show expression ratios of specific genes in wild-type plants under desiccation stress or control conditions (WT-D/C) and depict the induction of these genes in response to the stress treatment. With the exception of MYB29 and PAP2/MYB90, all of the gene are responsive to stress treatment in wild-type plants with expression levels increasing from twofold to threefold in several cases to nearly 70-fold with DREB2C and Xero2. The third bars in the graphs shown in Fig. 4 represent the
expression ratios between 35S::AtSAP5 plants exposed to desiccation stress versus plants of the same genotype under control conditions (OE-D/C), while the fourth bars represent the expression ratios between desiccation stress treated 35S::AtSAP5 plants and wild-type plants. These comparisons allow for estimation of the relative effects of AtSAP5 over-expression and stress treatment on the expression of specific genes. For example, expression of AtGolS2 in wild-type plants was strongly induced by desiccation (*20-fold) but induction associated with AtSAP5 overexpression was more modest (*twofold). However stress responsive expression in the 35S::AtSAP5 plants was more robust than in wild-type plants (*35-fold) indicating that the effects of AtSAP5 over-expression and stress are combinatorial. Similar responses were seen for SDR, UGT75B1 and UGT73B2. As second expression pattern is represented by ATCP1, DFR, and DREB2C. While expression of these genes was up-regulated in response to AtSAP5 over-expression under control conditions, desiccation treatment strongly induced their expression in both 35S::AtSAP5 plants and in control plants to similar levels. Expression of PAP1/MYB75 followed a similar pattern except that expression was more responsive to AtSAP5
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Table 1 Genes up-regulated in 35S::AtSAP5 plants under normal conditions that were analyzed by quantitative real-time PCR Accession no
Expression ratioa
Name
Description
At1g56650
PAP1/MYB75
MYB domain—containing transcription factor
2.86
At1g66390
MYB90/PAP2
Myb-related transcription factor
2.90
Transcription factor
2.01
Transcription factors
At1g30500 UDP-Glucosyltransferases At1g05680
Putative indole-3-acetate beta-glucosyltransferase
2.97
At4g34135
UGT73B2
Glucosyltransferase -like protein
2.87
At1g05560
UGT75B1
UDP-glucose:indole-3-acetate beta-D-glucosyltransferase
2.40
At2g15480
UGT73B5
Putative glucosyltransferase
2.29
At2g36790
UGT73C6
At2g36970 Glutathione transferases
Putative glucosyltransferase
2.24
Putative glucosyltransferase
2.11
At1g17170
ATGSTU24
Putative glutathione transferase
2.56
At2g29490
ATGSTU1
Putative glutathione S-transferase
2.12
At2g29420
ATGSTU7
Putative glutathione S-transferase
2.01 3.46
Dehydrogenases/reductases At5g42800
DFR
Dihydroflavonol 4-reductase
At1g76690
OPR2
12-oxophytodienoate reductase (OPR2)
2.79
At4g13180
SDR
Short-chain alcohol dehydrogenase
2.74
At3g29250
SDR
Short-chain alcohol dehydrogenase
2.41
At1g56600
AtGolS2
Water stress-induced protein, Galactinol synthase 2
3.55
At5g49480
ATCP1
NaCl-inducible Ca2?-binding protein
1.95
Stress
a
Expression fold increase in 35S::AtSAP5 plants relative to wild-type plants
over-expression than to desiccation treatment, while PAP2/ MYB90 was strongly induced in 35S::AtSAP5 plants but did not respond to the stress treatment. Finally, expression of DREB2A and Xero2 was strongly induced by desiccation stress but these genes were not responsive to AtSAP5 overexpression. It should be noted that the dehydrin gene Xero2 is regulated by DREB2A (Sakuma et al. 2006). Leaf stomata of AtSAP5 over-expressing plants were more closed than those of wild-type plants, resulting in reduced water loss through transpiration. While this could indicate that these plants are more sensitive to ABA than wild-type plants or have altered ABA metabolism, changes in the ABA sensitivity of germinating seeds or of leaf stomata were not detected (see Supplemental Fig 3 in Online Resource 1). Furthermore, changes in expression of ABA responsive or ABA biosynthetic genes were not seen in microarray experiments. AtSAP5 encodes a protein with two zinc finger domains that is targeted to the nucleus Structural analysis of the AtSAP5 gene shows that it consists of a single exon encoding a protein of 160 amino acids with a predicted molecular mass of 17.5 kDa (Fig. 5a).
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Analysis of the secondary structure of this protein identified two conserved domains, an A20 zinc finger domain located in the N-terminus at amino acids 26–39 and an AN1 zinc finger domain located in the C-terminus at amino acids 101–133. Sequence similarity searches revealed significant homology with other well-known A20-like proteins including stress associated protein 1 from rice (OSISAP1) (Mukhopadhyay et al. 2004), human zinc finger protein ZNF216, which is involved in NF-jB activation (Huang et al. 2004), associated with PRK1 protein (AWP1) (Duan et al. 2000), and human Rabex-5 (Mattera et al. 2006) (Fig. 5b). With the exception of Rabex-5, these proteins, showed strong sequence similarity within both the N-terminal A20 zinc finger motif, which contains the four conserved cysteine residues found in most A20 proteins, and a C-terminal domain, that contains the AN1 zinc finger motif (CX2–4CX9–12CX2CX4CX2HX5HXC) (Fig. 5c). On the other hand, the ubiquitin ligase Rabex-5, which does not contain AN-1 domain, has similarity only to the N-terminal A20 zinc finger motif of AtSAP5. Recent reports have shown that A20/AN1-like proteins can be localized to various cellular compartments (Duan et al. 2000; Hishiya et al. 2006; Kanneganti and Gupta 2008). The PSORT program (http://psort.ims.u-tokyo.ac.jp/
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DFR
ATCP1
AtGolS2
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4
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OE-C/WT-C WT-D/C
OE-D/C OE-D/WT-D
Fig. 4 AtSAP5 regulates the expression of stress responsive genes. Transcript levels of stress-responsive genes were determined by qRTPCT using RNA isolated from 2-week-old plants under either control conditions or following dehydration for 4 h. White bars represent expression ratios of the indicated gene expression in 35S::AtSAP5 transgenic plants compared to wild-type plants under control conditions (OE-C/WT-C). Grey bars indicate show expression ratios of
genes regulated after 4 h dehydration treatment compared to control conditions in wild-type plants (WT-D/C). Black bars show expression ratios of genes regulated after 4 h dehydration compared to control conditions in 35S:AtSAP5 transgenic plants (OE-D/C) and slashed bars show expression ratios in 35S::AtSAP5 and wild-type under dehydration conditions OE-D/WT-D. Error bars indicate SD, n = 3
form.html) was used to predict the subcellular localization of AtSAP5. This analysis indicated the presence of a putative bipartite nuclear targeting signature similar to the
nuclear-localization sequence (NLS) of nucleoplasmin (Efthymiadis et al. 1997) (Fig. 5a). To determine the subcellular localization of AtSAP5, it was fused in frame to
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Fig. 5 The AtSAP5 sequence and sequence alignment of A20-like proteins. a The sequence of AtSAP5 and its translated amino acid sequence. A putative bipartite nuclear localization sequence is underlined. b Comparison of conserved Cys residues at N-terminal A20 zinc finger domain from AtSAP5 and other A20-like proteins. Conserved amino acid residues are indicated in red. Number shown on right side of domain indicates a position of amino acid residues.
c Conservative Cys and His residues at C-terminal AN1 zinc finger domain are indicated in yellow. Number shown on right side of domain indicates a position of amino acid residues. OSISAP1 (stress associated protein 1, rice), A3C039; hZNF216 (ubiquitin binding protein, human), AAC61801; hAWP1 (PRK-1 associated protein, human), CAC14874; Rabex-5 (human Rab5 GDP/GTP exchange factor), Q9JM13
the C-terminus of green fluorescent protein (GFP) and transgenic plants that express the GFP-AtSAP5 fusion protein were observed by confocal microscopy. The fusion protein was targeted to the nuclei in root tips and elongation zone, indicating that AtSAP5 is nuclear localized (Fig. 6).
(Fig. 7b). Furthermore, GST-AtSAP5 undergoes autoubiquitination with addition of a single ubiquitin moiety detected in in vitro assays (Fig. 7a, lower panel). E3 ubiquitin ligases interact with a specific E2 ubiquitin conjugating enzymes in vivo to mediate ubiquitination (Hochstrasser 1996). Therefore, five recombinant human E2 enzymes, UbcH2, UbcH5a, Ubc5b, Ubc5c, and UbcH8 were tested for their ability to support AtSAP5-dependent ubiquitination in vitro. In these assays, AtSAP5 showed a clear preference for UbcH2 compared to other E2 enzymes in catalyzing poly-ubiquitin chain assembly (Fig. 7c). Since the structure of the N-terminal zinc finger domain in AtSAP5 is related to the cognate domain of other A20like proteins (Fig. 5b), we hypothesized that the A20 zinc finger domain of AtSAP5 was responsible for its E3 ligase activity. This was tested by a domain mapping experiment in which in vitro ubiquitination assays were performed using GST-fusion proteins that include either the A20 zinc finger domain or the AN1 zinc finger domain, with or without their flanking regions (Fig. 8a). The ubiquitin ligase activity of GST fusion proteins that contained either the A20 zinc finger domain alone (26–50) or the A20 zinc finger domain plus flanking regions (1–84) was reduced relative to the full length protein (Fig. 8b). The detection in
Arabidopsis SAP5 is an E3 ubiquitin ligase Several A20 zinc finger domain-containing proteins from animal cells act as E3 ubiquitin ligases (Mattera et al. 2006; Wertz et al. 2004). To determine if AtSAP has similar activity, recombinant AtSAP5 was produced and tested for E3 ubiquitin ligase activity in vitro. AtSAP5 was fused with an N-terminal GST tag, expressed in E. coli and the recombinant protein was purified by GSH affinity chromatography. In the presence of ubiquitin (Ub), yeast E1, and human E2, the GST-AtSAP5 fusion protein catalyzed the formation of poly-ubiquitin chains (Fig. 7a, upper panel lane5). No poly-ubiquitin forms were detected in reactions that lacked Ub, E1, E2, or GST-AtSAP5, indicating that AtSAP5 has E3 ubiquitin ligase activity (Fig. 7a, upper panel). Poly-ubiquitin chains were detectable within 15 min of incubation with GST-AtSAP5
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Fig. 6 Subcellular localization of GFP-AtSAP5 fusion protein. Fiveday-old T2 seedlings expressing the GFP-AtSAP5 fusion protein grown in MS agar plants in the light were analyzed for GFP expression using confocal microscopy. GFP fluorescence (a, d),
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counter stained for cell walls with propidium iodide (b, e), and overlay images (c, f). Images show root tip (a, b and c) and elongation zone (d, e and f). Bars 50 lm
Fig. 7 E3 ligase activity of the AtSAP5. a In vitro selfubiquitination reactions were performed without individual components (-Ub, -yeast E1, -E2 UbcH2, and -purified GST-AtSAP5) at 30°C for 2 h. Ubiquitin conjugates were resolved by 10% SDS–PAGE and detected by immuoblot analysis using anti-HA (top) or anti-GST (bottom). b GSTAtSAP5 was incubated at 30°C as indicated time in the presence of Ub, E1, E2 enzyme (UbcH2), and analyzed using immunoblots probed with the anti-HA antibody. *Indicate non specific binding. c Indicated various E2 enzymes assayed for ubiquitin-conjugating activity in the presence of GST-AtSAP5
these assays of ubiquitinated species with apparent molecular masses near to that estimated for the fusion protein itself appears to indicate that these A20-zinc finger domain proteins may only be capable of auto-ubiquitination in vitro. On the other hand, the GST fusion proteins that contained the AN1 zinc finger motif, either alone
(91–138) or in combination with its flanking sequences (58–160) exhibited E3 ubiquitin ligase activity that was comparable to the full length fusion protein (Fig. 8b). Thus, the AN1 zinc finger domain of AtSAP5 appears to be sufficient for full E3 ligase activity in vitro, while the A20 domain may only be capable of auto-ubiquitination under
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Fig. 8 AN1 zinc finger domain of AtSAP5 can catalyze E3 ligase activity. a Diagram of AtSAP5 protein and its truncated mutants used for E3 ligase activity assay. b In vitro self-ubiquitination assay using wild-type GST-AtSAP5 and GST-AtSAP5 truncated mutants (10% SDS–PAGE). *Indicates non-specific binding
these conditions, as indicated by immunoblot assays using anti GST (see Supplemental Fig 5 in Online Resource 1).
Discussion SAP5, a member of Stress Associated protein family in Arabidopsis thaliana, was chosen for analysis because of its sequence similarity to rice SAP1, a gene previously shown to be involved in stress responses (Mukhopadhyay et al. 2004). According to public microarray data, four of the 14 SAP genes in Arabidopsis, including SAP5, are induced by abiotic stress treatments (Zimmermann et al. 2004). Several investigations of the physiological functions of SAP genes have been reported (Huang et al. 2008; Kanneganti and Gupta 2008); however, information about the mode of action of SAPs is quite limited. To investigate the function of AtSAP5, Arabidopsis plants that express a 35S::AtSAP5 transgene were generated and their phenotypes compared with wild-type and atsap5 T-DNA knockdown plants under dehydration, osmotic and salt stress conditions. The results indicated that elevated expression of AtSAP5 increases tolerance to these stress treatments (Figs. 2, 3). 35S:AtSAP5 transgenic plants were less sensitive to osmotic and salt stress during germination and post-germinative growth. Although root
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growth of T-DNA knockdown seedlings was somewhat more sensitive than wild-type seedlings to osmotic and salt stress, the effect was not severe (Fig. 2c). This relatively weak phenotype could be due to the presence of the T-DNA insertions within the promoter region of AtSAP5, resulting in only a partial reduction in AtSAP5 expression. It is also possible that other SAP homologues can compensate for the reduction of SAP5 expression in these lines. Since T-DNA insertion mutants within the exon of SAP5 are not available, it may be necessary to use an RNAibased approach to further investigate the role of SAP5 in stress tolerance. SAPs that encode A20/AN1 zinc finger domain proteins are widely expressed in plants. These proteins contain a conserved zinc finger domain similar to those first identified in the mammalian A20 protein which plays a critical role in the inhibition of tumor necrosis factor (TNF)induced programmed cell death (De Valck et al. 1996; He and Ting 2002; Heyninck et al. 1999; Huang et al. 2004; Opipari et al. 1992). A20 contains seven of these zinc finger domains, but only the fourth domain (Znf4) is required for E3 ubiquitin ligase activity (Wertz et al. 2004). The single A20-like zinc finger domain of Rabex-5 is most similar to the fourth zinc finger domain of A20 and this protein also has E3 ubiquitin ligase activity (Mattera et al. 2006). Although the enzymatic activity of plant A20/AN1
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like proteins has not been previously reported, based on its sequence similarity with mammalian A20-like proteins (Fig. 5b), Kanneganti and Gupta (2008) suggested that AtSAPs may have E3 ubiquitin ligase activity. Data presented here indicates that AtSAP5 does have E3 ubiquitin ligase activity and undergoes auto-ubiquitination in vitro (Fig. 7a). Since AtSAP5 contains both A20-like and AN1like zinc finger motifs, we assayed each domain separately to determine their relative contributions to the E3 ubiquitin ligase activity of this protein. Interestingly, recombinant proteins the include the AN1-like zinc finger domain of AtSAP5 had E3 ubiquitin ligase activity comparable to the full length protein in vitro, while those that contained the A20-like zinc finger domain were only capable of autoubiquitination (Fig. 8 and see Supplemental Fig 5 in Online Resource 1). A20-like zinc finger domains typically contain glycine followed by paired aromatic amino acids after the first cysteine pair. Penengo et al. (2006) reported that the presence of tyrosine as the first aromatic amino acid of the pair in the Rabex-5 A20-like motif is critical for ubiquitin binding. Likewise, the catalytically active Znf4 domain of A20 has tyrosine at this position, while the inactive A20 Znf domains do not. On the other hand, ZNF216, which does not have detectable E3 ligase activity, has phenylalanine at this position (Hishiya et al. 2006; Huang et al. 2004). Phenylalanine is also present at corresponding positions in most Arabidopsis SAP proteins that have A20like domains (AtSAP1-AtSAP9) and in OSISAP1, while the A20-like zinc finger domain of AtSAP5, which lacks both aromatic amino acids, contains valine at this position and leucine is present in AtSAP10. This sequence divergence may help to explain the failure of A20 zinc finger domain of AtSAP5 to show significant E3 ubiquitin ligase activity and suggests that the A20 domains of other AtSAP proteins may have characteristics similar to ZNF216. The AN1 domain of AtSAP5 has E3 ubiquitin ligase activity in vitro (Fig. 8). Although the AN1 domains of AtSAP5 and ZNF216 are highly conserved (Fig. 5c), Hishiya et al. (2006) were unable to detect E3 activity with ZNF216. However, these authors concede that ZNF216 could have E3 ubiquitin ligase activity under assay conditions different than those used in their experiments. Little else is known about the biochemical function of AN1 zinc finger domains. AN1 zinc finger domains are similar to RING-H2 or RING variants in which the cysteine and histidine residues within the RING finger domain are swapped. For example, when compared to the consensus RING-H2 finger (C-X2-C-X(9-39)-C-X(1-3)-H-X(2-3)-HX2-C-X(4-48)-C-X(2)-C), cys6 and cys7 of the AtSAP5 AN1 zinc finger domain are replaced by histidine, while his4 and his5 are replaced by cysteine. Furthermore, the tertiary structure of the RING finger domain consists of
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two b-sheets, an a-helix, and two loops, while the predicted conformation of the AN1 zinc finger domain has two b-sheets, an incomplete a-helix and two loops (see Supplemental Fig 4 in Online Resource 1). The E3 ubiquitin ligase AvrPtoB, a protein with no apparent sequence similarity to RING finger or RING variant motifs was found to contain a RING domain fold in its three-dimensional crystal structure (Janjusevic et al. 2006). Thus, in spite of its sequence differences, it is possible that AN1 zinc finger domains can adopt a RING finger conformation and this may be responsible for the ubiquitin ligase activity of AtSAP5. There is ample evidence that the ubiquitination system is involved in regulating stress-responsive signaling pathways. For example, PUB22 and PUB23 ubiquitinate RPN12a, a part of the 26S proteasome complex, and pub22/pub23 mutant plants showed increased tolerance to drought stress (Cho et al. 2008). Interestingly, many E3 ubiquitin ligases act as negative regulators of stress responses. For example, HOS1 negatively regulates the expression of cold-responsive genes by ubiquitinating ICE1 (Dong et al. 2006), and DRIP1 targets ubiquitination of DREB2A, resulting in its destabilization and down regulation of stress responses (Qin et al. 2008). While AtCHIP is reported to mono-ubiquitinate the A subunit of PP2A and increase its activity, Arabidopsis plants that over-express AtCHIP plants showed increased sensitivity to cold stress (Yan et al. 2003). A novel E3 ligase, KEG was recently found to be regulated by ABA (Liu and Stone 2010; Stone et al. 2006). KEG interacts with and degrades ABI5, a positive regulator of ABA signaling. ABA promotes KEG degradation by self-ubiquitination and maintains a balance between KEG and ABI5. In addition, several E3 ligases have been shown to act as positive regulators of abiotic stress. These proteins include RHA2 (Bu et al. 2009), AtAIRP1 (Ryu et al. 2010), and SDIR1 (Zhang et al. 2007), which are involved in several aspects of ABA signaling. Proteins targeted by RHA2A, are proposed to be negative regulators of the ABA signaling pathway. Over-expression of AtAIPR1 leads to ABA hypersensitivity during seed germination and stomatal closure, resulting in tolerance to severe drought stress. Like SDIR1, AtAIPR1 up-regulates expression of genes that encode bZIP transcription factors and a number of other genes involved in ABA signaling. Ubiquitin ligases involved in regulation of ABA-independent pathways could include Rma1H1. Expression of Rma1H1 is not responsive to ABA and over-expression Rma1H1 in Arabidopsis confers tolerance to water stress by decreasing levels of the plasma membrane aquaporin, PIP2;1 via the 26S proteasome (Lee et al. 2009). Our data indicates that AtSAP5 could also act as a positive regulator of stress responsive pathways.
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ABA plays an important role in the regulation of plant stress responses (Shinozaki and Yamaguchi-Shinozaki 2000), resulting in stomatal closure to reduce water loss and elevated expression of genes involved in stress acclimation. Recent studies showed that increased expression of genes encoding the RING-domain proteins XERICO (Ko et al. 2006) or SDIR1 (Zhang et al. 2007) confer dehydration tolerance through ABA dependent pathways. Unlike SAP5, over-expression of XERICO leads to increased sensitivity to exogenous ABA and to salt and osmotic stresses during germination. Levels of ABA were also substantially increased in XERICO over-expressing plants, compared to wild-type plants, and expression of AtNCED3, an important ABA-biosynthetic gene, was induced more quickly in response to stress treatments. Over-expression of SDIR1 also led to ABA and salt hypersensitivity during germination and stomatal closure and drought tolerance in mature plants. While expression of AtSAP5 was strongly induced in response to exogenous ABA treatment, no significant differences in seed germination and seedling growth on media containing ABA were seen between wild-type, 35S::AtSAP5, and atsap5 plants and stomata of both AtSAP5 over-expressing plants and plants with reduced AtSAP5 expression retain ABA responsiveness (see Supplemental Fig 3 in Online Resource 1). Expression of the ABA-independent stress responsive transcription factor DREB2A (Liu et al. 1998) and its target gene, Xero2, (a dehydrin) was not affected under either control or dehydration treatments in 35S::AtSAP5 transgenic plants (Fig. 4). Interestingly, expression of DREB2C was induced in 35S::AtSAP5 transgenic plants under both control and dehydration conditions. DREB2C is induced by heat and salt stress treatments (Lim et al. 2007) and recent evidence indicates that DREB2C physically interacts with the ABA-dependent transcription factor ABF2, providing for potential crosstalk between the ABA-dependent and ABA-independent stress signaling pathways (Lee et al. 2010). Thus, if future experiments confirm that AtSAP5 is involved in the stress-responsive expression of DREB2C, it could indicate that AtSAP5 plays a role in mediating crosstalk between ABA-dependent and ABA-independent stress responsive signaling pathways. In conclusion, the results presented here indicate that elevated expression of AtSAP5 resulted in alterations in stress responsive gene expression that led to increase tolerance to osmotic stress in seedlings and water deficit in mature plants. In addition, our study shows that AtSAP5, a member of A20/AN1 type zinc finger protein, has E3 ubiquitin ligase activity in vitro and this activity is mediated primarily through the AN1 zinc finger motif. Future identification of putative targets of AtSAP5 ubiquitination should provide more definitive evidence for the function of
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this protein in plant cells and structural analysis AtSAP5 will be necessary to determine how these domains coordinate ubiquitination of target proteins. Acknowledgments The authors thank Dr. Yuhong Tang and Mr. Stacy Allen at the Noble Foundation Microarray Facility for microarray analyses and Ms. Karen Flowers at the Noble Foundation Greenhouse Facility for assistance with plant production. This work was funded in part by grants from the Oklahoma Center for the Advancement of Science & Technology and start-up funds from the Noble Foundation and the Walter Sitlington Foundation.
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