The Plant Journal (2010) 63, 179–188
doi: 10.1111/j.1365-313X.2010.04233.x
A HECT E3 ubiquitin ligase negatively regulates Arabidopsis leaf senescence through degradation of the transcription factor WRKY53 Ying Miao† and Ulrike Zentgraf* Center for Plant Molecular Biology (ZMBP), Department of General Genetics, University of Tu¨bingen, Auf der Morgenstelle 28, 72076 Tu¨bingen, Germany Received 2 March 2010; accepted 7 April 2010; published online 11 May 2010. * For correspondence (fax +49 7071 295042; e-mail
[email protected]). † Present address: Botanical Institute, University of Kiel, Olshausenstrasse 40, 24098 Kiel, Germany.
SUMMARY WRKY transcription factors play a central role in controlling leaf senescence in Arabidopsis. One important member, WRKY53, is tightly regulated by various mechanisms, and is a convergence node between senescence and pathogen responses. Using WRKY53 in a yeast two-hybrid screen, we isolated the HECT domain E3 ubiquitin ligase UPL5. In contrast to mammals, Arabidopsis contains only seven HECT E3 ubiquitin ligases, whose targets and functions are largely unknown. In yeast cells, UPL5 interacts with WRKY53 via its leucine zipper domain, and this interaction was confirmed in the cytoplasm of plant cells by a bimolecular fluorescence complementation assay. UPL5 was able to use the WRKY53 protein as a substrate for polyubiquitination in an in vitro system, and induction of UPL5 expression by an ethanol-inducible system in upl5 plants led to degradation of the WRKY53 protein. Expression of both genes is regulated antagonistically in response to hydrogen peroxide, jasmonic acid and plant development. Two T-DNA insertion lines (upl5-1 and upl5-2) showed the same senescence phenotype as WRKY53 over-expressers. Over-expression of WRKY53 in the upl5 background enhanced the accelerated senescence phenotype of WRKY53 over-expressers. Therefore, we conclude that UPL5 regulates leaf senescence in Arabidopsis through degradation of WRKY53 and ensures that senescence is executed in the correct time frame. Keywords: leaf senescence, HECT E3 ubiquitin ligase, protein degradation, WRKY transcription factor, hydrogen peroxide, jasmonic acid.
INTRODUCTION Senescence is a specific form of programmed cell death (PCD) that leads to the death of whole organs, e.g. leaves or flowers, and eventually to the death of entire plants. Like all forms of PCD, senescence is a highly regulated and energyconsuming process. However, in contrast to other forms of PCD, the prior aim of senescence is the mobilization of carbon, nitrogen and minerals out of the senescing tissue into developing parts of the plant. Many agriculturally important traits are affected by senescence processes, such as the number and quality of seeds, timing of seed set, fruit ripening, shelf life, etc. Despite the importance of the senescence processes in plants, our knowledge of the regulatory mechanisms of senescence is still fragmentary. The massive changes in the transcriptome imply an important role of transcription factors (Buchanan-Wollaston et al., 2003, 2005; Guo et al., 2004; Zentgraf et al., 2004; Balazadeh et al., 2008). Many transcription factors are ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd
themselves induced at the transcriptional level during leaf senescence (Chen et al., 2002; Buchanan-Wollaston et al., 2003, 2005; Guo et al., 2004). Expression profiling revealed that many WRKY factors are strongly up-regulated, and that WRKY factors constitute the second largest group of transcription factors in the senescence transcriptome (Guo et al., 2004). During dark-induced senescence, 21 of 59 WRKY factors are induced (Lin and Wu, 2004). However, the biological function of the individual WRKY factors expressed during leaf senescence is still unclear (U¨lker and Somssich, 2004). WRKY70 negatively regulates developmental senescence, whereas WRKY53 is a positive regulator (Miao et al., 2004; U¨lker et al., 2007). Target gene analyses of WRKY53 revealed that WRKY53 acts upstream of many other WRKY factors, but WRKY factors act in a regulatory network influencing the transcription of each other rather than in a linear signal transduction pathway (Eulgem et al., 2000; 179
180 Ying Miao and Ulrike Zentgraf Robatzek and Somssich, 2002; Dong et al., 2003; Li et al., 2004; Miao et al., 2004). Apart from auto-regulation and networking between WRKY factors, almost nothing is known about the upstream elements and factors controlling senescence-specific expression of the WRKY transcription factors. Recently, it was shown that epigenetic programming via histone methylation at WRKY53 locus controls leaf senescence in Arabidopsis (Ay et al., 2009). In addition, at least three proteins have been isolated that bind to the promoter of WRKY53. The activation domain protein (AD protein) has some similarity to histidine phosphotransfer (HPT) kinases, and works as an activator to up-regulate WRKY53 gene expression (Miao et al., 2008). In contrast, the singlestranded DNA-binding protein Whirly1 works as a repressor of WRKY53 expression (Ying Miao, Rena Isemer, Anke Scha¨fer, Nils Grabe, Kirsten Krause and Karin Krupinska, Botanical Institute, University of Kiel, unpublished results). Surprisingly, a mitogen-activated protein kinase kinase kinase (MEKK1) was also identified as a WRKY53 promoterbinding protein, and most likely regulates a switch from leaf age-dependent to plant age-dependent expression (Hinderhofer and Zentgraf, 2001; Miao et al., 2007). In addition, MEKK1 can directly phosphorylate the WRKY53 protein, increasing its DNA-binding activity (Miao et al., 2007). Moreover, our previous work showed that the WRKY53 protein interacts with a jasmonic acid-inducible protein. The gene of this interacting partner (ESR/ESP) encodes an epithiospecifier. These proteins can drive the conversion of glucosinolates into nitriles rather than isothiocyanates and are involved in pathogen defense. Expression of WRKY53 and ESR/ESP is antagonistically regulated in response to jasmonic and salicylic acid, and WRKY53 affects ESR/ESP expression and vice versa (Miao and Zentgraf, 2007). Therefore, the WRKY53 and ESR/ESP interaction appears to mediate negative cross-talk between pathogen defense and senescence, which is most likely governed by the jasmonic and salicylic acid equilibrium (Miao and Zentgraf, 2007). Here we report that WRKY53 can interact with one of the HECT domain E3 ubiquitin ligase proteins, UPL5, and is ubiquitinated by UPL5 in vitro. The induction of UPL5 expression by an ethanol-inducible system in upl5 plants clearly targets WRKY53 protein for degradation. Therefore, the WRKY53-mediated senescence pathway is regulated by WRKY53 protein modification and degradation. RESULTS WRKY53 and UPL5 interact in vivo in yeast and plant cells Using the yeast two-hybrid system, interacting partners of WRKY53 have already been characterized as influencing the activity of the protein, positively by phosphorylation (MEKK1) and negatively by blocking DNA binding (ESR/ESP) (Miao and Zentgraf, 2007; Miao et al., 2007). Another interacting protein that was isolated in the same screen encodes
a member of the HECT ubiquitin–protein ligase (UPL) family (Figure 1a,b). This family comprises seven members in the Arabidopsis genome, all containing the conserved approximately 350 amino acid HECT domain at the C-terminal end. These proteins can be grouped into four sub-families (UPL1/ 2, UPL3/4, UPL5 and UPL6/7) (Huibregtse et al., 1995; Downes et al., 2003). We identified UPL5 (At4g12570) 12 times in our screen. We isolated the full-length cDNA of UPL5 and cloned it into the yeast two-hybrid vector. The interaction was confirmed in the yeast two-hybrid system using this full-length construct either by growth on selection media or by X-Gal overlay assay (Figure 1a,c). In the next step, we deleted various parts encoding various protein domains (Figure 1b) from the yeast construct and quantified the b-galactosidase activity, which is only present if an interaction between UPL5 and WRKY53 takes place. The HECT domain, UBL domain and the C-type lectin binding domain of UPL5 appear to have only minor effects, but the interaction between UPL5 and WRKY53 is disrupted if the leucine zipper domain of UPL5 is deleted (Figure 1c). In order to characterize the function of UPL5 in more detail in planta, we first localized the protein by GFP fusion. The plasmid containing the fusion construct was transiently transformed into onion epidermal cells using the biolistic bombardment method (Krause et al., 2005). GFP-dependent fluorescence was analyzed 24 h after bombardment using epifluorescence or a confocal laser scanning microscope. Using these GFP fusion constructs, UPL5–GFP was found predominantly in the cytoplasm whereas WRKY53–GFP was predominantly localized in the nucleus, as expected (Figure 1d). The in planta interaction between UPL5 and WRKY53 was determined using bimolecular fluorescence complementation assays (BiFC). For this purpose, constructs in which the interacting proteins were fused to either the C-terminal (GFPc155) or the N-terminal (GFPn173) part of the GFP protein were made and transformed into plant cells. If the WRKY53 and the UPL5 proteins are able to interact directly in plant cells, the N-terminal and C-terminal parts of GFP will be brought into close proximity, and should emit green fluorescence. Using transformed onion cells, an interaction could be detected predominantly in the cytoplasm, exactly matching the localization of UPL5 (Figure 1d). To confirm this result in the homologous system, Arabidopsis protoplasts were prepared from leaves of wild-type plants and were transiently co-transformed with the 35S:GFPc155-HA-UPL5 and 35S:GFPn173-c-myc-WRKY53 plasmids. Whereas cells transformed with empty vectors or empty vector and 35S:GFPc155-HA-UPL5 produced no fluorescence, a strong signal was observed in the cytoplasm when GFPc155-HAUPL5 was co-expressed with GFPn173-c-myc-WRKY53 (Figure 1e). This clearly confirms an interaction of WRKY53 with UPL5 in the cytoplasm of Arabidopsis cells. In addition, other WRKYs (WRKY4, WRKY15 and WRKY33) were tested
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
E3 ubiquitin ligase regulates senescence 181 W53 UPL5 W53 vector vector UPL5 W53 vector UPL5 vector
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Figure 1. Interaction between WRKY53 and UPL5. (a) Yeast two-hybrid interaction test on selective and non-selective media combined with an X-Gal overlay assay. W53, WRKY53 full-length cDNA; UPL5, UPL5 full-length cDNA; vector, empty yeast vector. (b) Schematic drawing of the UPL5 protein. UBL, ubiquitin-like domain, most likely involved in interaction with other components of the ubiquitin system; C-type lectin, C-type lectin-binding domain, most likely involved in recognition of sugar moieties; LZ, leucine zipper domain for protein–protein interactions; HECT, a conserved 350-amino acid domain that is called the HECT domain based on its homology with the C-terminus of human E6-associated protein, a ligase that assists in the degradation of p53 (Huibregtse et al., 1995). The numbers indicate the amino acid residues. (c) Yeast two-hybrid interaction test with deletion variants of UPL5 on selective and non-selective media and quantification of the b-galactosidase activity. Error bars indicate the standard errors of four independent experiments. (d) Localization of UPL5 and WRKY53 GFP fusion proteins and a biomolecular fluorescence complementation assay in onion epidermal cells that were transiently transformed using particle bombardment. Transformed constructs are indicated on the right. Left, green fluorescence; middle, bright field; right, overlay. (e) Biomolecular fluorescence complementation assay using Arabidopsis protoplasts. Transformed constructs are indicated on the right. An empty vector was used as control (35S:GFPn173). Left, green fluorescence; middle, bright field; right, ethidium bromide staining of the nucleus.
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et al., 2004), but is also responsive to chinitin, Pseudomonas infection and salicylic acid (Dong et al., 2003). WRKY33 is also a group II WRKY factor and is induced by a variety of abiotic stresses such as salt, cold, heat, drought and osmotic stress (Jiang and Deyholos, 2009), and also by pathogen infection. In addition, WRKY33 is activated through MAP kinase signaling via MPK4 (Qiu et al., 2008). WRKY4 belongs to group I and is also involved in pathogen resistance (Lai et al., 2008), but has not been characterized as a direct target gene of WRKY53. All three genes respond to salicylic acid but show a maximum at different time points, WRKY4 after 1 h, WRKY33 after 4 h, and WRKY15 after 8 h (Dong et al., 2003). None of these factors showed interaction with UPL5 as visualized by green fluorescence using BiFC, although expression of the fusion proteins was detected on Western blots (data not shown). In vitro ubiquitination
(e) 35S:GFPn173-W53 35S:GFPc155-UPL5
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for their interaction with UPL5 using BiFC. These factors were chosen to test the participation of UPL5 in leaf senescence and also in processes like stress response and pathogen defense. All three factors are expressed during leaf senescence, but WRKY4 and WRKY15 are expressed to a much greater extent than WRKY33. WRKY15 is a group II WRKY factor and is a direct target gene of WRKY53 (Miao
In order to test whether the WRKY53 protein can be marked for degradation by the 26S proteasome through polyubiquitination by interaction with UPL5, we incubated recombinant His-tagged WRKY53 protein in the presence of recombinant GST-tagged UPL5 protein, E1 ubiquitinactivating enzyme, E2 ubiquitin-conjugating enzymes and ubiquitin. Subsequently, we analyzed the proteins on immunoblots using various antibodies. If all necessary components were added to the reaction, the anti-histidine antibody recognized some polyubiquitinated His-tagged WRKY53 proteins (Figure 2). If the conserved cysteine in the active site of the HECT domain of UPL5 was changed to a serine, resulting in a mutated version of UPL5 (Huibregtse et al., 1995), no ubiquitinated proteins were detected (Figure 2). This indicates that UPL5 has ubiquitin ligase activity
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
182 Ying Miao and Ulrike Zentgraf
Figure 2. Ubiquitination assay. Immunodetection of Western blots after an in vitro ubiquitination assay using recombinant proteins as indicated in the table: W53, 6 · His-tagged WRKY53; Ubi, ubiquitin; UPL, GST–UPL5 E3 ligase; UPLm, GST–UPL5 E3 ligase mutated at the conserved cysteine of the active site; E1, ubiquitin-activating enzyme E1 of rabbit; E2, ubiquitin-conjugating enzyme E2 of rabbit; Ubn-His-W53, polyubiquitinated 6 · His-tagged WRKY53. The antibodies used are indicated on the images.
in vitro and that WRKY53 can be used as a substrate in vitro. However, these in vitro tests appear to be very non-specific with respect to the target proteins (Yang et al., 2006), therefore this test was used to characterize the enzymatic function of UPL5 as an E3 ubiquitin ligase than to identify specific targets of UPL5. Degradation of WRKY53 by UPL5 in planta In order to determine whether UPL5 is involved in WRKY53 degradation in planta, the UPL5 full-length cDNA fused to an
HA tag was brought under the control of an ethanol-inducible promoter, and was transformed together with the 35S:cmyc-WRKY53 construct into upl5-1. The selected double transgenic plants were sprayed with ethanol, and the induction of expression of HA-tagged UPL5 protein and degradation of c-myc-tagged WRKY53 protein were analyzed by immunoblots using anti-HA or anti-c-myc antibodies. Ethanol treatment by spraying the transgenic plants with 1% ethanol led to the expression of UPL5 after 2 h (a faint band can already be detected on the Western blot after 1 h). In agreement, the amount of WRKY53 protein was clearly reduced after 2, 4 and 6 h, indicating that UPL5 is involved in the degradation of WRKY53 and most likely has ubiquitin ligase activity in planta (Figure 3). In contrast, ethanol treatment of upl5/35S:c-myc-WRKY53 without inducible UPL5 did not show any visible WRKY53 degradation. WRKY53 and UPL5 are antagonistically expressed during development and after hydrogen peroxide and jasmonic acid treatment WRKY53 shows a very characteristic expression pattern, switching from leaf age-dependent expression with high expression in old leaves to plant age-dependent high systemic expression during bolting (Hinderhofer and Zentgraf, 2001). This is exactly the time point at which senescence is turned on in all rosette leaves to mobilize carbon, nitrogen and mineral sources out of the senescing tissue into the developing flowers and seeds. Analyses of transgenic plants expressing the GUS gene under the control of the UPL5 promoter revealed that UPL5 is highly expressed in leaf tissue but expression is reduced at bolting when WRKY53 protein levels should be high (Figure 4a) (Hinderhofer and Zentgraf, 2001; Miao et al., 2004). Hydrogen peroxide
Figure 3. Degradation of WRKY53 by UPL5 in planta. Immunodetection of the tagged recombinant proteins in protein extracts isolated from double transgenic upl5 plants that over-express c-myc-tagged WRKY53 (35S:c-myc-WRKY53) and contain the UPL5 gene under the control of an ethanol-inducible promoter (AlcA:HA-UPL5). The various time points after 1% ethanol treatment are indicated above the lanes. Coomassie staining of an identical gel or Ponceau staining of the membrane were used as loading control for protein amounts. Transgenic plants expressing 35S:c-myc-WRKY53 in the upl5 background without the inducible UPL5 gene were also treated with 1% ethanol as a control.
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
E3 ubiquitin ligase regulates senescence 183
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Figure 4. Expression analyses of UPL5. (a) Rosettes of transgenic plants containing the UPL5 promoter-driven GUS gene were stained for GUS activity. The age of the plants is indicated in weeks. (b) Quantitative RT-PCR analyses of RNA isolated from 6-week-old plants sprayed with hydrogen peroxide (500 mM) or jasmonic acid (0.08 mM). ACTIN2 was used as an internal control. The values at the start of the treatment (0 h) were set to 1.0. Error bars represent standard deviations of three technical replicates of two biological replicates (n = 6).
measurements revealed that the hydrogen peroxide content in leaves increased exactly at this time point (Miao et al., 2004; Zimmermann et al., 2006). It was also shown that expression of WRKY53 as well as the expression of its upstream regulators can be induced by hydrogen peroxide (Miao et al., 2004, 2007, 2008). Unlike WRKY53, UPL5 expression is reduced after hydrogen peroxide treatment, as shown by quantitative RT-PCR (Figure 4b). UPL5 is induced by jasmonic acid treatment, but WRKY53 expression is reduced (Figure 4b), as shown previously (Miao and Zentgraf, 2007). Taken together, these data show that WRKY53 and
Senescence phenotype of upl5 plants If UPL5 targets a senescence-regulating transcription factor for degradation, we would expect that UPL5 knockout plants (upl5-1 and upl5-2) would have a senescence phenotype that is very similar to that of WRKY53-over-expressing plants (W53-OE). Therefore, two T-DNA insertion lines were characterized by a PCR screen, and loss of expression was tested by RT-PCR (Figure S1). Comparing the corresponding leaves of 7.5-week-old plants of W53-OE and upl5 with those of wild-type plants using a color code (Hinderhofer and Zentgraf, 2001), senescence appears to be accelerated in both plants to more or less the same extent. Two examples of each line are presented in Figure 5(a). As there were small differences between plants of the same lines (Figure 5a), a statistical analysis of 4–6 plants was performed by categorizing the leaves into four groups according to their leaf color (green; green/yellow; fully yellow; brown/dry; Figure 5b). Using this categorization, upl5-1, upl5-2 and W53-OE plants exhibited the same phenotypic appearance from 5.5 to 8 weeks of development. Moreover, leaves 6 and 7 of three 7.0-week-old plants were pooled, and expression of SAG12 was analyzed by quantitative RT-PCR using ACTIN2 as a reference (Figure 5c). The amount of SAG12 mRNA (as a marker for senescence) was higher in W53-OE, upl5-1 and upl5-2 than in wild-type plants. If WRKY53 was overexpressed in the upl5 background (Figure S1), senescence was even more accelerated in independent transgenic lines (Figure 5b,c). This clearly indicates that UPL5 most likely targets WRKY53 for degradation and is therefore involved in senescence regulation. Taken together, we were able to identify a direct target protein for a plant HECT ubiquitin E3 ligase and assign a biological function to this degradation process. DISCUSSION Arabidopsis WRKY proteins comprise a family of plant zincfinger-type transcription factors involved in the regulation of gene expression during pathogen defense, wounding, trichome development and senescence (Eulgem et al., 2000; U¨lker and Somssich, 2004). However, the individual role of the various WRKY factors is still unclear. WRKY53 regulates the expression of several senescence-associated genes (SAGs) and many other transcription factors (Miao et al., 2004). WKRY53 expression is switched from a leaf agedependent to a high systemic plant age-dependent pattern, most likely to induce senescence of the whole rosette during bolting (Hinderhofer and Zentgraf, 2001). This switch appears to be regulated by MEKK1, which can directly bind to the promoter of WRKY53. As MEKK1 has no transcriptional activation potential, the action of MEKK1 on the promoter of
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
184 Ying Miao and Ulrike Zentgraf
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Figure 5. Senescence-related phenotype. (a) Phenotype of the rosette leaves of 7.5-week-old plants arranged according to their age. (b) Leaves of 4–6 plants of each line were categorized into four groups according to their color (green; green/yellow; fully yellow; brown/dry), and the percentages of each group with respect to total leaf numbers are presented. Error bars indicate standard deviations. (c) Quantitative RT-PCR analyses of RNA isolated from leaves 6 and 7 of various plant lines using SAG12-specific primers. ACTIN2 was used as an internal control. W53-OE, WRKY53 over-expressing plants; upl5-1 and upl5-2, UPL5 knockout plants; upl5 W53-OE, WRKY53 over-expressing plants in the upl5 background (lines 6, 7 and 8); Col-0, wild-type plants. Error bars indicate standard deviations of three experiments.
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
E3 ubiquitin ligase regulates senescence 185 WRKY53 is most likely transmitted by other WRKY53 promoter binding proteins. A good candidate is a regulatory protein (AD protein), which binds to the WRKY53 promoter and can also interact with MEKK1 at the protein level (Miao et al., 2008). However, this protein cannot be phosphorylated by MEKK1 in vitro. WRKY53 itself can also bind to its own promoter and is also able to interact with MEKK1 on protein level. In this case, WRKY53 DNA-binding activity can be regulated by MEKK1 through phosphorylation (Miao et al., 2007). Recently, an epigenetic reprogramming via histone methylation was observed at the WRKY53 locus during senescence (Ay et al., 2009). An RNA-directed DNA methylation protein mutant (rdm4) also altered the WRKY53 transcription level (He et al., 2009). Moreover, WRKY53 activity is negatively regulated by interaction with ESR/ESP, an epithiospecifying protein. The expression of WRKY53 and ESR/ ESP is governed by the equilibrium of salicylic and jasmonic acid, and is also influenced by the presence of the proteins themselves, each negatively regulating the expression of the other by a so far unknown mechanism (Miao and Zentgraf, 2007). This indicates that the presence and activity of WRKY53 are very tightly regulated on several levels. In this paper, we show that degradation of WRKY53 is also regulated. The HECT domain E3 ubiquitin ligase UPL5 directly interacts with WRKY53 in the cytoplasm of plant cells. If UPL5 protein production was induced by an ethanolinducible system in upl5 plants, the amount of WRKY53 protein decreased significantly. This indicates that UPL5 appears to be directly involved in the degradation of WRKY53. Probably, as a kind of double bottom to the upand down-regulation of WRKY53 expression, the WRKY53 protein level is also controlled by UPL5, which itself is antagonistically expressed to WRKY53 with respect to development, hydrogen peroxide and jasmonic acid. However, expression of UPL5 is not changed dramatically according to our expression analyses and Genevestigator data (https://www.genevestigator.ethz.ch) indicating that UPL5 protein is permanently present to ensure that the amount of WRKY53 and/or other substrates remains low, except during bolting. We assume from the cytoplasmic localization of UPL5 that WRKY53 is marked for degradation directly after translation in the cytoplasm. When the WRKY53 protein translation rate is low, UPL5 will directly ubiquitinate WRKY53 and thereby prepare the protein for degradation by the 26S proteasome. As soon as the concentration of WRKY53 protein increases, it can escape UPL5 and can be transported into the nucleus to induce transcription of senescence-associated genes. Another possibility is that WRKY53 shuttles between the nucleus and the cytoplasm and is degraded in the cytoplasm by UPL5. The acceleration of senescence in upl5 plants and the enhancing effect of WRKY53 over-expression in the upl5 background support this model. Whether UPL5 exclusively targets WRKY53 for degradation or whether other WRKY
transcription factors are also degraded by this pathway is subject to further investigations, but WRKY4, WRKY15 and WRKY33 at least cannot interact with UPL5 in planta. However, nothing is yet known about other substrates of UPL5. UPL5 appears to have a unique characteristic in the UPL protein family due to the presence of the C-type lectinbinding domain. It also forms its own clade in an unrooted phylogenic tree if only the C-terminal HECT domain was used for the analyses. In this case, UPL1/2, UPL3/4 and UPL6/7 did not cluster with each other but instead clustered with HECT domain proteins of other organisms based on the protein domains preceding the HECT domain. These domains appear to be important for target specificity (Downes et al., 2003). The presence of the C-type lectinbinding domain in UPL5 suggests that glycosylated proteins are the targets of UPL5. Therefore, we looked for possible glycosylation sites in the WRKY53 protein. By computer analyses, two possible sites, N72 and N256, were detected. When these sites were mutated by site-directed mutagenesis to encode asparagine instead of glycine, the mutated form of WRKY53 (W53G) still interacted with UPL5 in the yeast system (data not shown). Whether the interaction would be more effective with a glycosylated form of WRKY53 and whether WRKY53 really is glycosylated in plant cells remains to be elucidated. The substrates for UPL1/2, UPL3/4 and UPL6/7 are still unknown. Based on their additional domains, interactions with targets bearing nuclear localization signals (UPL3/4) and calmodulin (UPL6/7) are predicted (Downes et al., 2003). UPL2 has been identified in the mitochondrial proteome, indicating a possible function in protein degradation of mitochondrial proteins (Heazlewood et al., 2004). For UPL3, a role in trichome development has been determined. Whereas wild-type Arabidopsis plants develop tri-radiated trichomes with restricted rounds of endoduplication (32C), the trichomes on upl3 plants often develop five or more branches, often undergoing an additional round of endoreplication resulting in some trichome cells with 64C. Therefore, UPL3 is most likely involved in degradation of a regulatory protein that normally promotes endoreplication and branching during trichome morphogenesis (Downes et al., 2003). UPL3 was shown to be identical to the product of the KAKTUS gene (El-Refy et al., 2003). However, the GA hypersensitivity of the upl3 mutants with respect to hypocotyl growth clearly demonstrates a role for UPL3 in addition to that in trichomes. As the phenotype of an upl4 mutant, the closest homolog of UPL3 in Arabidopsis, was different from that of upl3, the functions and targets of UPLs are distinct from each other in Arabidopsis. Similarly, we did not observe a trichome phenotype for our upl5 mutants, and upl3 mutants did not show a senescence phenotype (Downes et al., 2003). In contrast to Arabidopsis and yeast, which contain only a few HECT E3s, mice and humans have greatly amplified the
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
186 Ying Miao and Ulrike Zentgraf use of this ligase type, with 50 or more members in this gene family (Schwarz et al., 1998; Downes et al., 2003). Instead, Arabidopsis appears to have enlarged the gene groups of two other ligase types, the SCF complex and RING E3s, for selective ubiquitination, with more than 700 and 400 potential isoforms, respectively (Gagne et al., 2002; Kosarev et al., 2002). This kind of selective ubiquitination is also involved in regulation of leaf senescence in Arabidopsis. Leaves of the oresera9 (ore9) mutant of Arabidopsis exhibit increased longevity during age-dependent natural senescence. ORE9 was identified as a 693 amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1like 1), a component of the plant SCF complex, suggesting that ORE9 functions to limit leaf longevity through degradation of proteins that are required for delay of the leaf senescence program in Arabidopsis. However, the nature of these target proteins is still unknown (Woo et al., 2001). Recently, the NLA gene was identified as RING-type ubiquitin ligase that is involved in regulation of plant development, and the nla mutant failed to develop the essential adaptive responses to nitrogen limitation, but senesced much earlier and more rapidly than did the wild-type plants under insufficient inorganic nitrogen supply (Peng et al., 2007). Furthermore, the U-box E3 ubiquitin ligase SAUL1 prevents premature senescence by targeting Arabidopsis aldehyde oxidase 3 (AAO3) for ubiquitin-dependent degradation by the 26S proteasome (Raab et al., 2009). Taken together, various kinds of ubiquitin-mediated protein degradation appear to be involved in regulation of leaf senescence. One of the targets of this degradation appears to be the transcription factor WRKY53, which is degraded by the HECT domain E3 ubiquitin ligase UPL5. This specific WRKY factor appears to be very tightly regulated at various levels: (i) gene expression, (ii) protein activity, and (iii) protein degradation.
EXPERIMENTAL PROCEDURES Plant material Seeds of Arabidopsis thaliana, ecotype Columbia, were grown in a climatic chamber at 22C with 16 h of illumination under low light conditions (60 lmol sec)1 m)2). Under these conditions, plants developed flowers within 6–7 weeks, and mature seeds could be harvested after 10–12 weeks. Six-week-old plants were used for spraying with 500 mM H2O2 or 0.08 mM jasmonic acid. Two homozygous T-DNA insertion lines (SALK_116446 and SALK_114333) for UPL5 were characterized by PCR screening. SALK_116446 (upl5-1) was tested using the gene-specific primers 5¢-TGATCGGTCCAATTTGCTATC-3¢ and 5¢-ACTTGCTCCAAGATTGGTGTG-3¢ and the left border primer 5¢-TGGTTCACGTAGTGGGCCATCG-3¢ (LBa1 primer). SALK_114333 (upl5-2) was tested using the gene-specific primers 5¢-TGTCACGATTCCTTTACCACC-3¢ and 5¢-CGTTTTCCACTCGATTCTCTG-3¢ and the LBa1 primer. Expression of UPL5 in the homozygous lines was tested either by Northern blot (data not shown) or RT-PCR (Figure S1).
Transgenic plants For the UPL5:GUS construct, 1.5 kbp upstream of the UPL5 start codon were cloned into the pCB308 vector using BamHI and XbaI (Xiang et al., 1999). The UPL5 full-length cDNA was amplified and cloned from total RNA isolated from young leaves using RT-PCR. For the ethanol-inducible promoter construct, full-length cDNA with an HA tag was cloned into the pGTRV-binSRN vector under the control of the AlcA promoter (AlcA:HA-UPL5), including the constitutively expressed repressor of the AlcA gene (35S:alcR) and a Basta resistance gene (Syngenta, http://www.syngenta.com). This vector was transformed into the upl5-1 line (SALK_116446) together with the 35S:c-myc-WRKY53 construct described previously (Miao et al., 2004, 2007). All constructs were verified by sequencing. Arabidopsis transformation was performed by the vacuum infiltration procedure (Bechtold and Pelletier, 1998). The seeds of the transgenic plants were first selected on kanamycin plates and verified by PCR. These seedlings were further selected for the second construct by spraying with 0.1% Basta (Bayer Crop Science, http://www. bayercropscience.com/). Five-week-old transgenic plants were used for spraying with 1% ethanol to induce UPL5 expression under the control of an ethanol-inducible promoter. To avoid variability in expression levels of 35S:c-myc-WRKY53, leaves were taken from the same plant at different time points after treatment.
Quantitative RT-PCR RNA was extracted from Arabidopsis rosette leaves using a Gentra Purescript kit (Biozyme, http://www.biozym.com). First-strand cDNA was synthesized using the QuantiTect reverse transcription kit according to the manufacturer’s protocol (Qiagen, http://www. qiagen.com/). For quantitative RT-PCR, the cDNA samples were diluted with water (1:3). Gene-specific primers were developed for the genes WRKY53, UPL5 and SAG12, as well as for ACTIN2 as an internal control (ACTIN2 forward 5¢-AAGCTCTCCTTTGTTGCTGTT3¢, reverse 5¢-GACTTCTGGGCATCTGAATCT-3¢; WRKY53 forward 5¢-CAGACGGGGATGCTACGG-3¢, reverse 5¢-GGCGAGGCTAATGGTGGTG-3¢; UPL5 forward 5¢-TTAGGGTTCGTAGATTGGTGCT-3¢, reverse 5¢-TGTGCTCATCGGCATAAGTT-3¢; SAG12 forward 5¢-GCTTTGCCGGTTTCTGTTG-3¢, reverse 5¢-GTTTCCCTTTCTTTATTTGTGTTG-3¢). Expression of the genes was monitored using 0.5 ll of the diluted cDNA in a SYBR Green quantitative RT-PCR analysis using the QuantiFast SYBR Green PCR kit (Qiagen) according to the manufacturer’s protocol. Each reaction was repeated at least three times. Quantitative RT-PCR was initiated with denaturation at 95C for 5 min, followed by 40 cycles of denaturation at 95C for 10 sec and annealing/extension at 58C for 30 sec. To determine the specificity of the reaction, a melting curve analysis of the product was performed immediately after the final PCR cycle by increasing the temperature from 60 to 90C at 0.2C sec)1. PCR was performed using an Applied Biosystems 7300 real-time PCR system (http:// www.appliedbiosystems.com/), and data analysis was performed using the 7300 system software. Relative quantification of the expression levels was performed using the DDCT method as described by Pfaffl et al. (2001). Additionally, the end-time PCR products were separated on agarose gels to ensure amplification of a single specific product. Each value represents three technical replicates of two biological replicates (six measurements altogether).
Yeast two-hybrid system The yeast two-hybrid screen has been described previously (Miao and Zentgraf, 2007). For confirmation of the protein–protein interaction, the full-length UPL5 and WRKY53 cDNAs were cloned into the prey pGADT7 vector, which harbours the Leu2 selection marker,
ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 63, 179–188
E3 ubiquitin ligase regulates senescence 187 and the bait pGBKT7 vector, which contains the Trp1 selection marker, respectively. If WRKY53 was used as bait construct in pGBKT7, the truncated version of WRKY53 (without the activation domain) was inserted. The two-hybrid assays were performed as described in Clontech’s MATCHMAKER GAL4 two-hybrid system 3 protocol to confirm the interaction (http://www.clontech.com).
either anti-GST or anti-histidine monoclonal antibodies for 1 h (Sigma-Aldrich). Blots were washed in TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% TWEEN-20, pH 7.5) for 10 min (three times) before incubation with secondary antibodies. The blots were washed again with TBST for 10 min (three times), and secondary antibody conjugates were detected using a chemoluminescent substrate and exposed to X-ray films.
Bimolecular fluorescence complementation assay (BiFC) The full-length UPL5 cDNA with an HA tag was cloned into a pGFPc155 vector using PstI and sequenced. The full-length WRKY53 cDNA was cloned into a pGFPn173 vector as described previously (Miao and Zentgraf, 2007). Onion epidermal cells were transiently transformed using the biolistic bombardment method (Krause et al., 2005). GFP-dependent fluorescence was analyzed 24 h after transfection using an epifluorescence microscope (Axiophot, Zeiss, http://www.zeiss.com) and a confocal laser scanning microscope (Leica TCS LC1, http://www.leica-microsystems.com). The laser settings were as follows: 488 nm at 37% of maximal power and 543 nm at 100% of maximal power. Photomultiplicator PMT1 was set by using a 500–530 nm window to collect the GFP fluorescence. In addition, Arabidopsis protoplasts of wild-type plants were prepared and co-transformed with plasmids containing the constructs 35S:GFPn173-c-myc-WRKY53 and 35S:GFPn155-HA-UPL5 (Sheen, 2001; Miao and Zentgraf, 2007). After incubation for 36 h, transformed protoplasts were observed using a confocal laser scanning microscope, with settings as above. Ethidium bromide (1 lg ll)1) was used to stain the nuclei. Images were processed using the Adobe Photoshop software package (http://www.adobe.com/products/). WRKY4, WRKY15 and WRKY33 were used as controls in place of WRKY53. Proteins were isolated from the transformed tissue, separated on SDS–PAGE, blotted and immunodetected using anti-GFP antibodies (SigmaAldrich, http://www.sigmaaldrich. com).
Expression of recombinant proteins and Western blot analyses UPL5 and WRKY53 full-length cDNA were cloned into pGEX4T-2 or pQE expression vectors for production of recombinant proteins in Escherichia coli. The UPL5 mutant gene (C839S) was generated using a QuikChange site-directed mutagenesis kit (Stratagene, http://www.stratagene.com). All recombinant proteins were prepared according to the manufacturer’s protocols. Briefly, for purification of GST-tagged UPL5 proteins, BL21 bacteria were lysed in buffer containing 50 mM Tris/HCl (pH 7.4), 200 mM NaCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, 2 mM phenylmethylsulphonyl fluoride (PMSF) and complete proteinase inhibitor cocktail (Roche, http://www.roche.de), and the GST–UPL5 proteins were purified using a pre-Redi purification column (GE Healthcare, http:// www.gehealthcare.com/). The 6 · His-tagged WRKY53 protein was purified using nitrilotriacetate (NTA) resin (Qiagen). All proteins were dialyzed against dialysis buffer (20 mM Tris/HCl pH 7.4, 2 mM MgCl2, 150 mM NaCl, 2.5 mM 2-mercaptoethanol and 10% glycerol). To extract soluble proteins from plant tissue, 200 mg of leaf material were batch-frozen in liquid nitrogen, ground into powder, resuspended in 100 ll of extraction buffer (100 mM Tris, pH 7.2, 10% sucrose, 5 mM MgCl2, 5 mM EGTA, protease inhibitor), and centrifuged at 15 000 g for 10 min. The supernatant was used for immunoblot analysis. Proteins were separated on 6% acrylamide gels and transferred to nitrocellulose membranes using standard protocols. Membranes were blocked for 1 h at room temperature in TBS containing 5% w/v non-fat dry milk powder. The membranes were incubated with
Ubiquitination assay For the ubiquitination assay, each reaction contained 10 lg of recombinant ubiquitin (Sigma), 0.1 lg rabbit E1 and 0.22 lg E2 UbcH7 (both Boston Biochemicals, http://www.bostonbiochem. com), 400 ng purified GST–UPL5 or GST–UPL5(C839S) protein, 50 ng of purified 6 · His-tagged WRKY53, 2 mM ATP, 50 mM Tris/ HCl (pH 7.4), 5 mM MgCl2 and 2 mM DTT. After incubation at 30C for 2 h, the reaction was stopped with 4 · SDS–PAGE loading buffer at 95C for 5 min. A 10 ll aliquot of each reaction was analyzed by electrophoresis on 6% SDS–PAGE gels. Ubiquitinated WRKY53 proteins were detected by Western blotting using anti-histidine antibodies (Sigma-Aldrich).
Accession numbers The accession numbers for the sequences used in this paper are given in parentheses: ULP5 (At4g12570), UPL5 knockout line upl5-1, UPL5 knockout line upl5-2, WRKY53 (At4g23810), WRKY4 (At1g13960), WRKY15 (At2g23320), WRKY33 (At2g38470), SAG12 (At5g45890), ACTIN2 (At3g18780).
ACKNOWLEDGEMENTS We are grateful for the technical assistance of Dagmar Kolb (ZMBP, Department of Plant Biochemistry, University of Tu¨bingen) and Rena Isemer (Botanical Institute, University of Kiel). We thank the Nottingham Arabidopsis Stock Centre for supplying seeds of the UPL5 T-DNA insertion lines. The ethanol-inducible expression system was kindly provided by Syngenta. This work was financially supported by the Deutsche Forschungsgemeinschaft (SFB 446).
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Expression analyses of UPL5 and WRKY53 in transgenic lines. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
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