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First published online October 13, 2006; 10.1104/pp.106.087965 Plant Physiology 142:1664-1682 (2006) © 2006 American Society of Plant Biologists Heterologous Expression and Molecular and Cellular Characterization of CaPUB1 Encoding a Hot Pepper U-Box E3 Ubiquitin Ligase Homolog1,[C]Department of Biology, College of Science (S.K.C., H.S.C., M.Y.R., M.M.L., H.S.P., W.T.K.) and Protein Network Research Center (Y.-Y.B.), Yonsei University, Seoul 120–749, Korea; and Department of Plant Biotechnology/Agricultural Plant Stress Research Center, Chonnam National University, Gwangju 500–712, Korea (M.J.P., J.K.)
The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35S::CaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.
Higher plants are continuously faced with various environmental stresses during their entire life cycle. Drought, high salinity, heavy metals, and extreme temperatures are common abiotic stresses that seriously impair the growth and development of soil plants. Water deficiency resulting from dehydration and high salinity is one of the most severe environmental factors responsible for the reduction of crop yield on as much as one-half of the world's irrigated land (Boyer, 1982  ; Cushman and Bohnert, 2000). To cope with such unfavorable growth conditions, plants have developed unique defense mechanisms and processes for acclimation that enhance their tolerance to detrimental conditions. A number of genetic and cellular events that occur under such stress have been widely documented (Bray, 1997; Ishitani et al., 1997; Zhu, 2002). Diverse sets of genes induced by drought and/or salt stress have been identified recently with the aid of combined molecular and genetic approaches (Ingram and Bartels, 1996; Cushman and Bohnert, 2000; Seki et al., 2001, 2002; Ozturk et al., 2002; Oono et al., 2003). However, the biological functions of these genes in terms of stress tolerance or sensitivity are still largely unknown in higher plants. Thus, it is of immense importance to study the functions of stress-inducible genes to understand the molecular mechanisms of stress tolerance in crop plants.
The ubiquitin (Ub) 26S proteasome system is a crucial regulatory mechanism for protein degradation in all eukaryotic cells. This system degrades a wide range of proteins in the nucleus and cytoplasm and plays a key role in the control of cellular functions as diverse as cell cycle progression, endocytosis, protein sorting, embryogenesis, hormone responses, defense against pathogens, and senescence (Frugis and Chua, 2002
Hot pepper (Capsicum annuum L. cv Pukang), a solanaceous species closely related to tobacco (Nicotiana tabacum), is one of the most economically important crops and is cultivated widely in East Asia for its hot-tasting fruits. We are interested in elucidating the adaptive response of hot pepper plants against abiotic stresses, such as water deficit. Recently, we have isolated and characterized a broad spectrum of cDNAs from hot pepper plants whose expression is enhanced rapidly in response to drought (Choi et al., 2002
Isolation and Identification of Full-Length CaPUB1 cDNA
Using differential display PCR and subtractive hybridization analysis, we previously identified a broad spectrum of partial cDNA clones from hot pepper seedlings, which were rapidly induced by dehydration (Choi et al., 2002
CaPUB1 Possesses E3 Ub Ligase Activity in Vitro and the U-Box Motif Is Essential for Enzyme Activity
Many U-box-containing proteins function as E3 Ub ligases (Hatakeyama et al., 2001
Organization and Expression of the CaPUB1 Gene From the results described above, it appears that CaPUB1 is a U-box-containing E3 Ub ligase. We therefore wanted to characterize the CaPUB1 gene in more detail at the molecular level. To assess the exact copy number of CaPUB1 in the hot pepper genome, genomic Southern-blot analysis was carried out using pCaPUB1 as a probe. Genomic DNA isolated from mature leaves of hot pepper plants was digested with EcoRI or XbaI and hybridized with 32P-labeled pCaPUB1 under high-stringency conditions. This hybridization detected only one clear band (Fig. 3A ), implying that the CaPUB1 gene is present as a single copy in the haploid hot pepper genome.
To examine the spatial expression pattern of the CaPUB1 gene, we investigated the level of corresponding mRNA in different hot pepper vegetative tissues by RNA gel-blot analysis. Total RNA samples were isolated from leaves, stems, and roots of 2-week-old light-grown plants and hybridized with the 32P-labeled gene probe under high-stringency conditions. The results revealed that the amount of mRNA was very low in all tissues examined, with its level being barely detectable after prolonged exposure of the blot to x-ray film (data not shown). Because CaPUB1 was identified initially in water-stressed leaves, we considered the possibility that the expression of CaPUB1 is modulated by abiotic stresses. To test this possibility, its mRNA accumulation profile was monitored under various abiotic stress conditions. As a first step, 2-week-old light-grown hot pepper plants harvested from agar plates were dehydrated on Whatman 3MM filter paper at room temperature and approximately 60% humidity under dim light. The degree of water stress was determined by the decrease in the fresh weight of the plants. Figure 3B shows that the low, basal level of the transcript (approximately 1.5 kb) begins to elevate pronouncedly in response to 5% water loss. Although this increase was seen in every tissue examined, including leaves and roots, the highest induction was in the root tissue. Expression of CaPUB1 mRNA was further stimulated as the plants were exposed to more severe water loss (15%–20%). Next, the expression profile of CaPUB1 was examined under salt stress. Induction of the CaPUB1 transcript was clearly detected after a 2-h treatment of the root tissue with 200 mM NaCl (Fig. 3C). This marked increase in mRNA level was continuously maintained for at least 24 h. In contrast, expression of the gene was unaffected by high salinity in the leaves. This indicates that root tissue is a major part of the plant to induce the CaPUB1 gene in response to water and salt stresses. Likewise, cold temperature (24 h at 4°C) also enhanced CaPUB1 gene expression in root and leaf tissues, although the induction was less evident than with water or salt stresses (Fig. 3D). Thus, these results demonstrate that the CaPUB1 gene is activated in response to a broad spectrum of abiotic stresses. To further understand the regulation of CaPUB1, we analyzed its expression in response to mechanical wounding, one of the most common stresses that plants encounter during their life cycle. The CaPUB1 transcript in leaves was induced rapidly (within 30 min) upon wounding, attaining a maximal level at 1 h, and declining thereafter (Fig. 3E). A positive control for wound induction, the proteinase inhibitor II homolog, CaPINII, showed a distinct pattern of activation compared with CaPUB1. The transcript began accumulating at 1 h and increased continuously for at least 4 h after wounding. This indicated that the induction of CaPUB1 by wounding was not an artifact. Intriguingly, as was the case for CaPINII, slight activation of CaPUB1 was also observed in unwounded systemic leaves (Fig. 3E). On the other hand, expression of the CaPUB1 gene was unaffected by abscisic acid (ABA), ethylene, or the bacterial pathogen Xanthomonas axonopodis (data not shown). Overall, these results are consistent with the view that the CaPUB1 gene is subject to control by diverse environmental factors, but not by biotic stresses.
To address the cellular function of CaPUB1, attempts were made to establish transgenic hot pepper plants that constitutively expressed the CaPUB1 gene. Unfortunately, this approach turned out to be unsuccessful due to technical difficulties. Transformation and regeneration yield was extremely low so that we could not obtain enough independent transgenic lines. Instead, we established transgenic Arabidopsis plants that overexpressed CaPUB1 under the control of the cauliflower mosaic virus 35S constitutive promoter. Numerous independent T4 transgenic lines that exhibited markedly enhanced levels of the CaPUB1 transcript under normal growth conditions were chosen for further analysis (Fig. 4A ).
Figure 4, B and C, show the morphological comparison of 35S::CaPUB1 and wild-type seedlings at an early stage of development. During our search for phenotypic differences, we observed that most of the independent 35S::CaPUB1 seedlings displayed significantly longer hypocotyls and roots than the control seedlings under dark growth conditions, with the root length being predominantly different (Fig. 4B). Morphology of cotyledons, however, was somewhat similar in the dark. Under greenhouse conditions, root growth could be more readily distinguishable. We found that the roots of 35S::CaPUB1 seedlings were 1.6- to 1.7-fold longer than in control plants (Fig. 4C). A more unique phenotype specific for the 35S::CaPUB1 construct was that the transgenic lines grew more rapidly than the controls, thereby resulting in early bolting (Fig. 4D). Under our experimental conditions, the majority of wild-type Arabidopsis plants bolted at 29 to 30 d after imbibition. On the other hand, most independent CaPUB1-overexpressing plants exhibited earlier bolting at 26 to 27 d after imbibition (Fig. 4D). The detailed cellular phenotype was further investigated by comparing roots of 5-d-old light-grown 35S::CaPUB1 and wild-type Arabidopsis seedlings. Analysis of transverse sections showed that the organization of the cortex, endodermis, and stele cell layers of CaPUB1-overexpressing roots appeared to be abnormal. In most sections, transgenic roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers, as compared with the control roots (Fig. 5A ). Furthermore, discontinuity in the endodermis cell layer was found frequently, which we have not been able to observe in wild-type roots (Fig. 5A). The cortex cell layer also was discontinuous in some transgenic roots. Altered cell layers were also observed in the longitudinal sections of roots; additional cell layers were seen clearly around the endodermis and cortex in the transgenic lines (Fig. 5B).
Extra cells in the cortex, endodermis, and steles in the 35S::CaPUB1 transgenic lines probably arose from additional cell divisions. This hyperproliferative phenotype prompted us to examine the expression of cell cycle-controlled genes by reverse transcription (RT)-PCR. As shown in Figure 6A , the transcript level of the D-type cyclin CycD3 (Riou-Khamlichi et al., 1999 ), the cell cycle-dependent kinase-related gene CDC2b (Yoshizumi et al., 1999), the S-phase-specific proliferating cell nuclear antigen (PCNA), and the chromosome assembly histone H3 (Kosugi and Ohashi, 2003) was significantly up-regulated in CaPUB1-overexpressing lines 13 and 44. Thus, increased numbers of cells in 35S::CaPUB1 plants correlate with increased expression of cell cycle progression genes. Taken together, these results, along with those in Figures 4 and 5, led to the suggestion that, under these experimental conditions, CaPUB1 was functionally relevant in heterologous Arabidopsis cells and, hence, might participate in a subset of the positive control of cell and tissue growth in transgenic lines.
Overexpression of CaPUB1 Changes Plant Response to Water and Salt Stresses The aforementioned results concerning the RNA expression profile led us to hypothesize that the hot pepper CaPUB1 might function in the defense mechanism against abiotic stresses. Therefore, the effects of CaPUB1 overexpression on the response of Arabidopsis to water and salt stresses were examined. First, root and hypocotyl growth assays were performed with 5- to 7-d-old transgenic and wild-type seedlings that had been incubated with 50 to 100 mM NaCl. As illustrated in Figure 7 , significant differences were observed between the 35S::CaPUB1 and wild-type plants after being exposed to NaCl treatment. Whereas elongation of etiolated control seedlings was generally unaffected by the presence of 50 mM NaCl, growth of the dark-grown transgenic roots and hypocotyls was severely impaired by this mild salinity, indicating increased sensitivity to salt stress (Fig. 7A). Consequently, the clearly distinct phenotype of wild-type and 35S::CaPUB1 seedlings became quite similar in the presence of 50 to 100 mM NaCl (Fig. 7A). Under light conditions, the growth of control roots was enhanced slightly in the presence of 50 mM NaCl. In contrast, the light-grown 35S::CaPUB1 roots displayed a significant reduction of growth in response to mild salinity (Fig. 7B). This also reveals enhanced sensitivity to salinity. In the presence of 100 mM NaCl, elongation of wild-type roots was approximately 75% of normal, whereas elongation of transgenic roots was greatly reduced to 36% to 44% of the control, depending on the independent transgenic line (Fig. 7B).
High concentrations of salts inhibit germination of Arabidopsis (Quesada et al., 2000 ; Zhu, 2000). Under mild salt stress (10–100 mM NaCl), however, the germination efficiency of wild-type plants was only slightly affected (Fig. 7C). In contrast, germination of 35S::CaPUB1 plants was reduced 9% to 15% by 10 mM NaCl, 15% to 17% by 50 mM NaCl, and 22% to 24% by 100 mM NaCl, indicating that CaPUB1-overexpressing plants are hypersensitive to salt at the germination stage. Thus, both the germination (Fig. 7C) and postgermination (Fig. 7, A and B) growth of 35S::CaPUB1 transgenic plants are more sensitive to mild salinity than wild-type plants. On the other hand, wild-type and transgenic plants exhibited similar sensitivity to exogenously applied ABA (0.011 µM) in germination (Fig. 7C). This is in line with the result that expression of the CaPUB1 gene is not responsive to ABA, suggesting that the function of CaPUB1 might be independent of ABA.
In the next experiment, we addressed the capacity of wild-type and 35S::CaPUB1 plants to respond to dehydration. Dehydration sensitivity was scored as the capacity of plants to resume growth after water stress when returned to normal conditions. Three- or 4-week-old Arabidopsis plants were grown in pots. When the soil was allowed to dry by withholding water for 12 d, all plants displayed wilting. After rewatering for 3 d, 33 of 59 wild-type plants survived and continued to grow (55.9% survival; Fig. 8
). Under these experimental conditions, however, most of the CaPUB1-overexpressing lines examined were unable to recover after rewatering, as opposed to wild type, and eventually died (1.5%–9.1% survival; Fig. 8). In addition, the expression level of RD29a, a typical drought stress-induced gene (Liu et al., 1998
Two-Dimensional Gel Electrophoresis and Protein Identification by Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry in Transgenic Arabidopsis Plants
In vivo and in vitro results showed that hot pepper CaPUB1 was an E3 ligase and that perturbation of CaPUB1 expression resulted in changes in cell growth and differentiation and alteration of plant response to abiotic stress. This raised the question as to whether CaPUB1 exerted its cellular functions by participating in the mechanism by which certain proteins were degraded by the Ub 26S proteasome pathway. To unravel this question, we decided to employ two-dimensional gel electrophoresis (2-DE) analysis. Total proteins were extracted from 7-d-old whole seedlings of wild-type and 35S::CaPUB1 line 13 and subjected to a linear pH 4 to 7 immobilized pH gradient strip in the first dimension. Subsequently, 9% to 16% gradient SDS-PAGE was carried out in the second dimension, as described in "Materials and Methods." After electrophoresis, the resolved proteins were visualized by staining with silver nitrate and the number of individual protein spots on the gel was analyzed using Melanie III software (Fig. 9
). Individual proteins with Mr values from 20,000 to 70,000 and pIs from 4.5 to 5.5 and from 5.5 to 7.0, respectively, were analyzed because maximal separation and reproducibility were obtained within these parameters. Among the numerous silver-stained proteins, we detected a spot whose abundance was significantly reduced in 35S::CaPUB1 line 13 as compared to wild type (Fig. 9). This spot was excised from the gel, digested with trypsin, and then analyzed by matrix-assisted laser-desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS). The protein identified by this method turned out to be the 26S proteasome regulatory subunit RPN6 (Kwok et al., 1999
CaPUB1 Interacts with RPN6 Protein
RPN6 protein was identified initially as the Arabidopsis non-ATPase S9 (subunit 9) of the 19S regulatory particle (RP) from the 26S proteasome complex (Kwok et al., 1999 As a first step to investigate this possibility, immunoblot analysis was performed using an anti-RPN6 antibody in wild-type and transgenic plants. The results showed that the relative abundance of RPN6 protein decreased significantly in both transgenic lines 13 and 44 compared to that in wild-type plants (Fig. 10A ). To obtain evidence indicating a specific interaction between CaPUB1 and RPN6, we performed an in vitro pull-down assay. Both CaPUB1 and RPN6 were expressed in E. coli cells as MBP and haemagglutinin (HA) fusion proteins, respectively. The purified fusion proteins were coincubated with an amylose affinity matrix, followed by extensive washing. The bound protein was eluted from the amylose resin by 10 mM maltose and immunoblotted with the anti-HA antibody. Figure 10B shows that HA-RPN6 was pulled down from the amylose affinity resin by the MBP-CaPUB1 protein, but not by HA-RPN6 alone. As a specificity control, we tested the interaction between RPN6 and CaRDCP1, a hot pepper RING-finger domain Ub ligase. As shown in Figure 10B, CaRDCP1 failed to bind RPN6. These results suggest that the CaPUB1 and RPN6 proteins physically interact in vitro. In the next experiment, the in vitro ubiquitination assay was carried out. In this experiment, CaPUB1 was used as a source of E3 Ub ligase. The bacterially expressed HA-RPN6 protein was incubated at 30°C in the presence or absence of Ub, ATP, E1, E2, and MBP-CaPUB1 for the appropriate time period and subjected to immunoblotting using the anti-HA antibody. Figure 10C shows the production of higher molecular weight ubiquitinated ladders when HA-RPN6 was incubated with MBP-CaPUB1 in the presence of Ub, ATP, E1, and E2. The exclusion of any E1, E2, or MBP-CaPUB1 abolished the ubiquitination signals (Fig. 10C). Furthermore, the CaPUB1 T51A mutant failed to ubiquitinate HA-RPN6, suggesting that the ubiquitination of RPN6 by CaPUB1 was not an experimental artifact, but was due to the specific interaction between these two proteins (Fig. 10C).
To provide additional evidence for the interaction between CaPUB1 and RPN6, two in vivo experiments were performed. First, wild-type and 35S::CaPUB1 transgenic seedlings were treated with 100 µM cycloheximide for different time periods (0–3 h) in the absence or presence of MG132 (10 µM), an inhibitor of the 26S proteasome. During incubation, changes in the level of RPN6 protein were monitored using an anti-RPN6 antibody. As shown in Figure 10D, the amount of RPN6 protein was gradually decreased in the absence of MG132 in a time-dependent fashion, with the protein level being lower and slightly more rapidly degraded in the 35S::CaPUB1 seedlings compared to the wild type. In contrast, the protein level was unchanged when wild-type and transgenic seedlings were incubated with MG132. These results indicate that RPN6 was degraded in a proteasome-dependent manner in CaPUB1-overexpressing seedlings. Second, transgenic Arabidopsis plants expressing the CaPUB1-Flag fusion protein were constructed. The putative interacting proteins with CaPUB1 were immunoprecipitated using an anti-Flag antibody from the 35S-CaPUB1-Flag seedlings. The immunoprecipitated proteins were subsequently analyzed by immunoblotting with an anti-RPN antibody. The result in Figure 10E shows that RPN6 was immunoprecipitated by an anti-Flag antibody, indicating the in vivo interaction between CaPUB1-Flag and RPN6 proteins. This suggests that RPN6 indeed interacted with CaPUB1 and degraded in a CaPUB1-dependent manner in CaPUB1-overexpressing transgenic plants. Overall, the results in Figures 9 and 10 are consistent with the hypothesis that hot pepper CaPUB1 Ub ligase interacts with RPN6, thereby affecting the turnover of the RPN6 protein in 35S::CaPUB1 transgenic Arabidopsis plants.
The molecular and cellular processes underlying the acclimation of hot pepper to abiotic stresses have attracted much interest because it is an economically important crop and its response to adverse environmental factors is not well understood. From water-stressed hot pepper plants, we isolated the CaPUB1 gene that encodes a protein containing a single U-box motif, which is a highly conserved domain found in the diverse isoforms of eukaryotic E3 Ub ligase (Fig. 1). With the aid of in vitro ubiquitination and site-directed mutagenesis assays, we were able to confirm that bacterially expressed CaPUB1 possessed E3 Ub ligase activity and that the N-terminal U-box motif was essential for its enzyme activity (Fig. 2). RNA gel-blot studies showed that, in hot pepper seedlings, CaPUB1 mRNA was highly inducible in response to diverse environmental stresses, including dehydration, high salinity, and cold temperature (Fig. 3). It was worth noting that CaPUB1 was also rapidly activated (within 30 min) in both local and systemic leaves by mechanical wounding, although induction in systemic leaves was less pronounced than in local leaves. In contrast, CaPUB1 was not induced by ethylene, ABA, or infection by a bacterial pathogen. Thus, it would be reasonable to consider that, in hot pepper, CaPUB1 may not play a role in the biotic stress response, but rather functions specifically in early events in the abiotic-related defense response to deal with the effective plant adaptation process.
Although a number of U-box-containing proteins have been known to function as E3 Ub ligases (Hatakeyama et al., 2001
Nevertheless, we could not rule out the possibility that overexpression of a heterologous CaPUB1 gene with a strong cauliflower mosaic virus 35S promoter in Arabidopsis may create a pleiotropic phenotype that is difficult to interpret. In this regard, it is critical to consider the link between the cellular function of CaPUB1 and the disordered cell layers with small-sized cells and hypersensitivity to drought and salt in CaPUB1-overexpressing plants. We speculate on the possibility that the early bolting phenotype of the 35S::CaPUB1 plants, which results in early production of seeds, could be one of the possible mechanisms by which plants escape from drought or salt stress. Indeed, it was reported that there was a large positive genetic correlation between dehydration avoidance and flowering time (drought escape) among 39 different naturally occurring genotypes of Arabidopsis (Mckay et al., 2003
However, it should be noted that overexpression of CaPUB1 results in hypersensitivity to drought and salt. It has been known that the normal response of plants to abiotic stresses is growth arrest rather than promotion. Also, faster growth of vegetative organs does not necessarily result in early flowering. Thus, hypersensitivity to drought and salt and a rapid-growing phenotype of 35S::CaPUB1 plants could indicate that CaPUB1 probably functions as a negative regulator that inhibits the adaptive stress response. In this regard, the early-flowering phenotype of stressed plants might not be because of enhanced growth, but because of other flowering-time control mechanisms. Negative function of U-box E3 Ub ligase SPL11 has been known in the control of cell death and pathogen defense in rice plants (Zeng et al., 2004
High-resolution 2-DE has recently been shown to be a powerful tool for studying complex patterns of protein expression in various tissues of higher plants (Roberts, 2002
The 26S proteasome is a 2-MD ATP-dependent protease complex that removes many Ub-conjugated cellular proteins. The complex is composed of the 20S catalytic CP and two 19S RPs that form the base and lid at the ends of the CP (Voges et al., 1999 A database search identified that CaPUB1 shares significant sequence identity with two Arabidopsis U-box proteins, AtPUB22 (At3g52450; 51% amino acid identity) and AtPUB23 (At2g35930; 52% amino acid identity). Intriguingly, expression of both Arabidopsis U-box genes is highly inducible by various abiotic stresses (S.K. Cho, M.Y. Ryu, and W.T. Kim, unpublished data). Therefore, it is of immense importance to study cellular functions of these Arabidopsis CaPUB1 potential orthologs. In conclusion, the results presented in this study suggest that CaPUB1 acts in transgenic Arabidopsis plants to turn on a subset of physiological responses for coping with dehydration and high-salinity stresses. Further functional studies of the Arabidopsis PUB1 orthologs and characterization of the protein it interacts with will be required to further our understanding of the relationship between E3 Ub ligases and responses to abiotic stresses in higher plants.
Plant Materials and Stress Treatments
Dry hot pepper seeds (Capsicum annuum L. cv Pukang) were soaked once with 70% ethanol and then rinsed extensively with sterilized water. Seedlings were grown in a mixture of soil and vermiculite or on Murashige and Skoog medium containing 1% Suc, vitamin B5 (12 mg L–1), and 0.8% agar (pH 5.8) in a 25°C growth chamber with a 16-h light/8-h dark photoperiod. Hot pepper plants were subjected to various biotic and abiotic stresses as described by Park et al. (2003)
The full-length pCaPUB1 cDNA clone was introduced to the plasmid pMAL-c2X (New England BioLabs) and used for expression of the MBP-CaPUB1 fusion protein. The Escherichia coli BL21 (DE3) strain containing the constructed plasmid was grown at 37°C in 100 mL of Luria-Bertani medium (10 g tryptone, 5 g yeast extract, and 10 g L–1 NaCl) supplemented with 50 µg mL–1 ampicillin. Cells were grown for an additional 4 h at 30°C after induction with 1 mM isopropylthio-
The bacterially expressed MBP-CaPUB1 fusion protein (500 ng) was brought to 150 µL with ubiquitination reaction buffer (50 mM Tris-HCl, pH 7.5, 2.5 mM MgCl2, 0.5 mM dithiothreitol [DTT]) containing 4 mM ATP and 20 ng each of human E1 and E2 UbcH5B. The reaction mixture was incubated for the appropriate time periods (0–180 min) at 30°C. The reaction was stopped by the addition of SDS sample buffer. After boiling for 10 min, the sample was separated by 8% SDS-PAGE and subjected to immunoblotting using the anti-MBP antibody (New England BioLabs).
Hot pepper leaf genomic DNA was isolated as described previously (Park et al., 2003
Full-length pCaPUB1 cDNA was inserted into the corresponding sites of the binary vector pMBP2. The fusion gene construct was transferred to Agrobacterium tumefaciens strain AGL1 by electroporation, as described by Joo et al. (2004)
Assay of hypocotyl and root growth under salt stress was performed as described by Cho et al. (2006)
Wild-type and transgenic seeds collected at the same time were used. The germination ratio was monitored in the absence or presence of various concentrations of NaCl (10–100 mM) or ABA (0.01–1 µM; Cho et al., 2006
Five-day-old light-grown wild-type and transgenic Arabidopsis seedlings were fixed in PBS buffer containing 4% paraformaldehyde and embedded in plastic resin (Technovit 8100; Heraeus Kulzer) following the manufacturer's manual. Samples were cut into 2-µm thicknesses using a microtome (Leica Instruments). The sections were stained with a 0.1% aqueous solution of Toluidine Blue O (Sigma) for 1 min and visualized by light-field microscopy.
RT-PCR was performed in a total volume of 25 µL containing 1 µL of the first-strand cDNA reaction products, 1 µM primers, 10 mM Tris (pH 8.0), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 200 µM deoxynucleotides, and 2.5 units of Taq polymerase (Promega). Twenty-five thermal cycles were carried out, each consisting of 45 s at 95°C, 1 min at 60°C, and 90 s at 72°C in an automatic thermal cycler (Perkin-Elmer/Cetus). PCR products were separated on a 1% agarose gel and then visualized under UV light.
The 35S::CaPUB1 and wild-type Arabidopsis seedlings were harvested into lysis buffer containing 9 M urea, 4% CHAPS, and protease inhibitor cocktail (Roche Diagnostics). After homogenizing extensively for 5 min on ice, the cell lysates were centrifuged at 100,000 g for 15 min at 4°C in a Beckman TL-100 table ultracentrifuge. The supernatants were taken as the total protein extract and stored at –70°C until use. Protein extracts were normalized with a Bradford protein assay kit from Bio-Rad. Protein (350 µg) in 350 µL 2-DE sample buffer (9 M urea, 4% CHAPS, 100 mM DTT, 0.5% Pharmalyte [pH 4–7], and trace bromphenol blue) was loaded into 18-cm, pH 4 to 7 isoelectric focusing gel strips (Amersham-Pharmacia Biotech) by the in-gel rehydration method (Rabilloud et al., 1994
Protein spots on silver-stained gels were excised and digested with sequencing-grade trypsin, as described by Mang et al. (2004a)
For in vitro pull-down assay, the bacterially expressed MBP-CaPUB1 and HA-RPN6 fusion proteins were coincubated at 30°C for 30 min in a 750-µL reaction buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 1 mM EDTA), containing 30 µL amylose resin (New England BioLabs) with gentle agitation. The resin was washed three times with 500 µL reaction buffer. MBP-CaPUB1 was eluted from the amylose affinity resin with 150 µL buffer containing 10 mM maltose and then subjected to immunoblotting with anti-HA antibody (New England BioLabs). For in vivo pull-down assay, putative interacting proteins with CaPUB1 were pulled down by an anti-Flag antibody from 35S::CaPUB1-Flag transgenic seedlings. Pulled-down proteins were then analyzed by immunoblotting using an anti-RPN6 antibody.
Protein samples were separated by 12.5% SDS-PAGE and transferred to a nitrocellulose membrane as described (Lee et al., 2006 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number DQ211901. Received August 3, 2006; accepted October 5, 2006; published October 13, 2006.
1 This work was supported in part by grants from the Basic Research Program of the Korea Science and Engineering Foundation (project no. R01–2004–000–10487–0 to W.T.K. and H.S.P.), the Plant Diversity Research Center (Twenty-First Century Frontier Research Program funded by the Ministry of Science and Technology of the Korean government to W.T.K., H.S.P., and J.K.), and the Plant Metabolism Research Center at Kyung Hee University (Science Research Center project no. R11–2000–081 from the Korea Science and Engineering Foundation to W.T.K.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Woo Taek Kim (wtkim{at}yonsei.ac.kr).
[C] Some figures in this article are displayed in color online but in black and white in the print edition. www.plantphysiol.org/cgi/doi/10.1104/pp.106.087965 * Corresponding author; e-mail wtkim{at}yonsei.ac.kr; fax 82–2–312–5657.
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ABSTRACT
RESULTS
-uni-Zap II cDNA library constructed from water-stressed leaves of hot pepper plants. Numerous putative CaPUB cDNA clones were isolated. Restriction enzyme mapping and DNA-sequencing analysis indicated that these clones represented a single group of overlapping sequences. Among the isolated clones, pCaPUB1 contained the longest insert. The pCaPUB1 insert was 1,405 bp, consisting of a 95-bp 5'-untranslated region, a 1,221-bp coding region encoding 407 amino acids, and an 89-bp 3'-untranslated region (
I mutant protein. Taken together, it seems most likely that CaPUB1 possesses E3 Ub ligase enzyme activity. 
U-BOX is a deletion mutant in which the N-terminal U-box domain is truncated. D, In vitro ubiquitination assay of the wild-type and V 
-galactoside at OD600 0.6 to 1.0. The pellet was resuspended in phosphate-buffered saline (PBS) containing 1 mM phenylmethylsulfonyl fluoride. The suspension was sonicated on ice with a Vibracell sonicator (Sonics and Materials). Triton X-100 was added to a final concentration of 1% (v/v). The fusion protein was purified by affinity chromatography using MBP Sepharose 4B from MBP purification modules (Pharmacia), as described previously (Lee et al., 2004
-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid, and 50% acetonitrile, and analyzed with a PerSeptive Biosystem Voyager-DE STR MALDI-TOF mass spectrometer in reflector mode over a mass range of 1,000 to 3,000 D. Proteins were identified by peptide mass fingerprinting with the search programs ProFound (
