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The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in Arabidopsis  

The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in... The Plant Cell, Vol. 21: 3554–3566, November 2009, www.plantcell.org ã 2009 American Society of Plant Biologists The WRKY6 Transcription Factor Modulates PHOSPHATE1 W OA Expression in Response to Low Pi Stress in Arabidopsis 1 1 1 2 Yi-Fang Chen, Li-Qin Li, Qian Xu, You-Han Kong, Hui Wang, and Wei-Hua Wu State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, National Plant Gene Research Centre (Beijing), Beijing 100193, China Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and b-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression. INTRODUCTION PHO1 is predominantly expressed in the stellar cells of the root and the lower part of the hypocotyls and is believed have a role in Phosphorus (P), as a major essential nutrient for plant growth and Pi efflux out of root stellar cells for xylem loading (Hamburger development, serves various basic biological functions in the et al., 2002). However, PHO1 shares no homology with any plant life cycle (Raghothama, 1999). Phosphate (H PO ,orin 2 4 previously described Pi transporter proteins in plants and fungi short, Pi) is the major form that is most readily taken up and (Hamburger et al., 2002). It is interesting that PHO1 contains a transported in the plant cell (Ullrich-Eberius et al., 1981; Tu et al., SPX domain, which can be found in several proteins that are 1990). The Pi concentration in the soil, typically 10 mM or less, involved in phosphate transport and/or Pi signaling pathways in results in Pi starvation for plant growth and survival, which is one plants and yeast. For example, an SPX protein in yeast named of major limiting factors for crop production in the cultivated PHO81 is a key regulator in transporting and sensing phosphate, soils. A number of studies have shown that plants have evolved as well as in sorting proteins to endomembranes (Lenburg and different strategies to overcome limited Pi availability. In re- O’Shea, 1996; Wykoff and O’Shea, 2001). In Arabidopsis, the sponse to low Pi stress or Pi starvation, plants may increase the SPX proteins SPX1-SPX3 are involved in Pi signaling pathways Pi uptake from the soil by alteration of root architecture and and regulate the expression of the Pi transporter genes Pht1;4 function (Lo´ pez-Bucio et al., 2003; Ticconi and Abel, 2004; and Pht1;5 (Duan et al., 2008). Thus, the possibility cannot be Osmont et al., 2007). Under Pi-limiting conditions, plants may excluded that PHO1 may not be a direct Pi transporter but rather also increase their Pi acquisition by changing their metabolic and may regulate Pi loading of the xylem either by directly influencing developmental processes (Raghothama and Karthikeyan, 2005), the activity of transporter proteins or via signal transduction. such as increasing phosphatase activity (Lipton et al., 1987) and PHO1 gene expression can be induced by Pi starvation secretion of organic acids (Marschner, 1995). (Stefanovic et al., 2007; Ribot et al., 2008; also see Figure 5B in PHOSPHATE1 (PHO1) has been shown to play roles in Pi this study), but the transcription factors that regulate PHO1 translocation from root to shoot (Hamburger et al., 2002), which expression remain unknown. Transcriptome analysis has dem- is also important for plant adaptation to a low Pi environment. A onstrated that expression of many genes is significantly changed single nuclear recessive mutation in PHO1 led to its inability to in Oryza sativa (Wasaki et al., 2003) and Arabidopsis thaliana (Wu load Pi into xylem (Poirier et al., 1991; Hamburger et al., 2002). et al., 2003; Misson et al., 2005) under Pi-limiting conditions, indicating that transcriptional regulation may play important roles These authors contributed equally to this work. in plant responses to low Pi stress. More recently, a number of Address correspondence to wuwh@public3.bta.net.cn. regulatory components that may be involved in plant responses The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described to low Pi stress have been reported, such as microRNA miR339 in the Instructions for Authors (www.plantphysiol.org) is: Wei-Hua Wu (Bari et al., 2006; Chiou et al., 2006), Arabidopsis posttranslation (wuwh@public3.bta.net.cn). regulators PHOSPHATE TRANSPORTER TRAFFIC FACILITA- Online version contains Web-only data. OA TOR1 (At PHF1) (Gonza´ lez et al., 2005) and E3 SUMO Ligase Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.108.064980 (At SIZ1) (Miura et al., 2005), transcription factors PHOSPHATE WRKY6 Regulates PHO1 Expression 3555 STARVATION RESPONSE1 (At PHR1) (Rubio et al., 2001) and O. plants were grown in a potting soil mixture (Figure 1A). However, sativa Pi STARVATION-INDUCED TRANSCRIPTION FACTOR1 the overall growth of the WRKY6 null mutant wrky6-1,an En-1 (Os PTF1) (Yi et al., 2005), and Arabidopsis MYB62 transcription insertion mutant, was obviously better than wild-type plants factor (At MYB62) (Devaiah et al., 2009) and Arabidopsis (Figure 1A). The measured free Pi concentration in the potting WRKY75 transcription factor (At WRKY75) (Devaiah et al., soils was ;10 mM in this study. Thus, the plants grown under the 2007a). described conditions as shown in Figure 1A were actually WRKY proteins are plant-specific transcription factors encoded experienced low Pi stress. Under this low Pi stress condition, by a multigene family comprising 74 members in Arabidopsis the 35S:WRKY6-9 line displayed thinner stalks and smaller (http://www.Arabidopsis.org/browse/genefamily/WRKY-Som. leaves compared with wild-type plants (Figure 1A). Figure 1B jsp), and many of them have been found to play important roles in shows that the En-1 insertion in wrky6-1 disrupted the transcrip- plant responses to biotic and abiotic stresses. In addition to a tion of the WRKY6 gene. number of WRKY genes that have been demonstrated to be Besides the 35S:WRKY6-9 line, two more WRKY6-overex- involved in plant responses to pathogen infection and other pressing lines, Super:WRKY6-13 and Super:WRKY6-18, were defense-related stimuli (Dong et al., 2003; Kalde et al., 2003; Li included in our further experiments. The elevated expression of et al., 2004; Eulgem and Somssich, 2007), some WRKY genes WRKY6 mRNA in these transgenic lines is shown in Figure 1C. have also been shown to function in plant responses to various Transcription of WRKY6 in either wild-type or the wrky6-1 plants abiotic stress, such as drought (Pnueli et al., 2002; Rizhsky et al., was not detectable in our RNA gel blot experiments. Among the 2002; Seki et al., 2002; Mare, et al., 2004), cold (Huang and three WRKY6-overexpressing lines, 35S:WRKY6-9, Super: Duman, 2002; Seki et al., 2002; Mare, et al., 2004), heat (Rizhsky WRKY6-18, and Super:WRKY6-13 displayed the highest, me- et al., 2002), salinity (Seki et al., 2002), wounding (Hara et al., dium, and the lowest WRKY6 expression, respectively. 2000), and Pi starvation (Devaiah et al., 2007a). However, little is Plants usually accumulate more anthocyanin in their aerial known about the specific interaction of a given WRKY protein portions in response to low Pi stress (Marschner, 1995), and this with a defined target gene. Recent studies using an oligodeoxy- can result in brown-colored leaves. When grown on Murashige nucleotide decoy strategy have revealed that SUSIBA, a WRKY and Skoog (MS) medium with sufficient Pi supply, all tested protein, can bind to SURE (sugar responsive) and W-box ele- plants showed no difference in their phenotypes (top panel in ments in the iso1 promoter (Sun et al., 2003). Petroselinum Figure 1D). After the low Pi treatment, the WRKY6-overexpress- crispum WRKY1 has been shown to bind to the W-box of its ing plants, particularly Super:WRKY6-18 and 35S:WRKY6-9 native promoter as well as to that of Pc WRKY3 and Pc PR1-1 lines (both have much higher WRKY6 expression than does the based on chromatin immunoprecipitation (ChIP) analysis Super:WRKY6-13 line), displayed dark-brown leaves similar to (Turck et al., 2004). Candidate target genes for At WRKY53 the phenotype of the pho1 mutant (bottom panel in Figure 1D). To were identified with a pull-down assay (Miao et al., 2004), and confirm further the effects of WRKY6 overexpression on the low electrophoretic mobility shift assays identified candidate targets Pi response phenotype, another group of WRKY6-overexpress- for Hordeum vulgare WRKY transcription factor WRKY38 (Zou ing lines (35S:WRKY6-3, 35S:WRKY6-5, and 35S:WRKY6-9; et al., 2008), Arabidopsis WRKY26 and WRKY11 (Ciolkowski Robatzek and Somssich, 2002) were tested. As shown in Sup- et al., 2008), Nicotiana tabacum WRKY1, WRKY2, and WRKY4 plemental Figure 1 online, overexpression of WRKY6 indeed (Yamamoto et al., 2004), and O. sativa WRKY71 (Zhang et al., increased plant sensitivity to low Pi stress. Furthermore, increase 2004). of transgenic plant sensitivity to low Pi stress was closely related At WRKY6 was first reported to be associated with senes- to WRKY6 expression level (see Supplemental Figure 1 online). cence- and defense-related processes, and it could activate the A defect in Pi transfer from root to shoot has been reported in expression of its target gene SIRK, a receptor-like protein kinase the pho1 mutant (Poirier et al., 1991; Hamburger et al., 2002), in the process of senescence (Robatzek and Somssich, 2002). resulting in reduced Pi content in the shoot and smaller plant size. Here, we report a previously unknown function of WRKY6 in plant Under either Pi-sufficient or Pi-deficient conditions, the WRKY6- responses to low Pi stress. We demonstrate that plants over- overexpressing lines showed similar reduced Pi contents in expressing WRKY6 become more sensitive to low Pi stress and shoots as the pho1 mutant (Figures 2A and 2B). As a result, the display a similar phenotype as the pho1 mutant. WRKY6 nega- ratios of Pi content in shoot to that in root (Pi /Pi ) for both shoot root tively regulates PHO1 expression by binding to two W-box WRKY6-overexpressing lines and the pho1 mutant were signif- consensus motifs within the PHO1 promoter, and the repression icantly lower than the ratio determined in wild-type plants, of PHO1 expression by WRKY6 is released under low Pi condi- particularly under low Pi condition (Figures 2C and 2D). In tions. addition, four Super:PHO1 lines (Super:PHO1-1, -7, -9, and -13) with differential PHO1 expression were selected for the Pi content assay. As shown in Supplemental Figure 2 online, all Super:PHO1 lines and the wrky6-1 mutant displayed higher Pi RESULTS contents in shoots, whereas the pho1 mutant showed the lowest Pi content in shoots under both MS and low Pi (LP) conditions. WRKY6 Overexpression Plants Showed Similar Phenotypes The results demonstrate that Pi content in shoots indeed corre- as the pho1 Mutant under Low Pi Conditions lates with PHO1 expression. These data suggest that WRKY6 The growth of the aerial portion of the Arabidopsis WRKY6- may play a role in plant responses to Pi starvation at least partially overexpressing line (35S:WRKY6-9) was impaired when the through regulating PHO1-dependent Pi transfer. 3556 The Plant Cell It should be noted that the wrky6-1 mutants were obviously growing better than wild-type plants under the low Pi condition (Figures 1A and 1D, bottom panel) in our experiments, although they showed no difference under Pi-sufficient conditions (Figure 1D, top panel). However, Robatzek and Somssich (2001, 2002) had not ob- served phenotype difference between the wrky6-1 mutants and wild-type plants. After we have grown the plants under different environmental conditions, we believe that this difference mainly resulted from growth conditions, particularly light period. In the studies by Robatzek and Somssich (2001, 2002), plants were grown first under short-day conditions followed by long-day periods, while plants were grown under a constant long-day (18 h light) condition in our experiments. When Arabidopsis plants were grown under a short-day condition (10-h light), almost no phenotype difference between wild-type and wrky6-1 plants was observed (as shown in Supplemental Figure 3 online). WRKY6 Interacts with Two W-Box Motifs of the PHO1 Promoter To test the hypothesis that WRKY6 regulates PHO1 expression, we first tested whether WRKY6 could bind the PHO1 promoter. It is known that WRKY proteins usually bind to the W-box motifs of their target gene promoters (Eulgem et al., 2000). Analysis of the primary sequence of the PHO1 promoter revealed six W-box consensus motifs within the PHO1 promoter and four of them (named W ,W ,W , and W , respectively) are located at the very Q X Y Z end of promoter nearing the coding region (Figure 3A). The in vivo interaction between WRKY6 and the W-box motifs of the PHO1 promoter was investigated using the ChIP-qPCR (chromatin immunoprecipitation quantitative PCR) method. As shown in Figure 3B, WRKY6 strongly interacted with the PHO1 promoter when the primer combinations encompassing either W or W Y Z were applied, while no interaction was observed between WRKY6 and PHO1 promoter containing only W or W box. Q X These results demonstrated that WRKY6, as a transcription factor, can bind to two (W and W ) W-box motifs within the Y Z PHO1 promoter nearing the coding region, suggesting regulation of PHO1 transcription by WRKY6. WRKY6 Negatively Regulates PHO1 Transcription Based on the results of the phenotype tests (Figure 1), Pi content measurements (Figure 2), and ChIP analysis (Figure 3B), we further hypothesized that WRKY6 may negatively regulate PHO1 type seedlings. Seven-day-old seedlings were used for RNA extraction. EF1a was amplified for the control. Figure 1. Phenotype Tests of Various Plant Materials. (C) RNA gel blot analysis of WRKY6 expression in the WRKY6-over- (A) Phenotype comparison of the WRKY6-overexpressing line (35S: expressing lines (Super:WRKY6-13, Super:WRKY6-18,and 35S: WRKY6-9), the WRKY6 En-1 insertion mutant (wrky6-1), the pho1 mu- WRKY6-9) and the wrky6-1 mutant. Seven-day-old seedlings were tant, and wild-type (Columbia-0 [Col-0]) plants. All plants were grown in a used for RNAs extracted. The ethidium bromide–stained rRNA band potting soil mixture (rich soil:vermiculite = 2:1, v/v) and kept in growth was shown for the loading controls. 2 1 chambers at 228C with illumination at 120 mmol·m ·s for an 18-h daily (D) Phenotype comparison of the various plant lines as indicated. The light period for 30 d. 7-d-old seedlings germinated on MS medium were transferred to MS (B) RT-PCR test of WRKY6 expression in the wrky6-1 mutant and wild- (top panel) or LP (bottom panel) medium for another 7 d. WRKY6 Regulates PHO1 Expression 3557 Figure 2. Pi Content Measurements in Various Plant Materials. The 7-d-old seedlings of WRKY6-overexpressing lines (Super:WRKY6-13, Super:WRKY6-18, and 35S:WRKY6-9), the wrky6-1 mutant, the pho1 mutant, and wild-type plants germinated on MS medium were transferred to MS ([A] and [C])orLP([B] and [D]) medium for another 7 d, and then the shoots and roots of the seedlings were harvested separately for Pi content measurements. (A) and (B) Pi contents in roots and shoots of tested plant materials. Three replicates were included for each treatment, and experiments were repeated three times. Data are shown as means 6 SE (n =3). (C) and (D) Comparison of the ratio of Pi to Pi . The ratio was calculated from the data presented in (A) and (B).Dataare shownasmeans 6 SE (n =3). shoot root transcription. To test this hypothesis, we first compared the cating the removal of negative regulation (Figures 4B to 4E). On transcription of PHO1 in the roots of WRKY6-overexpressing the other hand, strong GUS expression was detected in wrky6-1 lines, wrky6-1, and wild-type plants, since both WRKY6 and roots regardless of which promoter fragment was used (Figures PHO1 are highly expressed in roots (Robatzek and Somssich, 4B to 4E). The GUS expression level was much higher in wrky6-1 2001; Hamburger et al., 2002). As shown in Figure 4A, the roots than in wild-type roots when the reporter gene was driven transcription of PHO1 in roots was repressed in the WRKY6- by promoters containing W and W (Figures 4B, 4C, and 4E). Y Z overexpressing lines. Repression of PHO1 expression was also However, there was almost no difference in the GUS staining closely related to the WRKY6 expression levels in WRKY6- among the roots of all three different types of plants when no overexpressing lines, with the strongest repression in 35S: W-box motif existed (Figures 4D and 4E). More importantly, in the WRKY6-9 plants and the weakest repression in Super: roots of 35:WRKY6-9 plants expressing the GUS reporter gene WRKY6-13 plants. driven by W - and W -containing promoter fragments, only weak Y Z We further tested if WRKY6 binding to PHO1 W-box motifs GUS staining can be detected (Figures 4B, 4C, and 4E). The was required for its function in regulation of PHO1 transcription. results demonstrate that binding to PHO1 W-box motifs was Different truncated PHO1 promoter fragments (indicated above required for WRKY6 regulation of PHO1 transcription. All these each panel of Figures 4B to 4D) driving the b-glucuronidase data support the notion that WRKY6 is the negative regulator of (GUS) reporter gene were transformed into 35S:WRKY6-9, PHO1 transcription. wrky6-1, and wild-type plants. In wild-type plants, the GUS reporter gene was expressed when driven by PHO1 promoter Repression of PHO1 Transcription by WRKY6 Is Removed fragment containing all four W-box motifs (W ,W ,W , and W ), Q X Y Z under Low Pi Stress two W-box motifs (W and W ), or no W-box motifs, respectively Y Z (Figures 4B to 4D). When all four W-box motifs were deleted from Consistent with previous reports (Stefanovic et al., 2007; Ribot the PHO1 promoter, the expression of the reporter gene in 35S: et al., 2008), we observed that PHO1 transcription was induced WRKY6-9 and wild-type roots was dramatically increased, indi- in response to low Pi stress. The PHO1 transcription level in 3558 The Plant Cell tion could be weakened. Protein gel blot analysis was performed using anti-WRKY6 serum in the total proteins extracted from the roots of seedlings grown on the low Pi medium. As shown in Figure 7A, the low Pi treatment induced a time-dependent decrease of WRKY6 protein content. Yeast two-hybrid assays (see Supplemental Table 1 online) showed that WRKY6 inter- acted with a RING-type finger E3 ligase (At1g74410), indicating that WRKY6 protein degradation may be mediated by the 26S proteosome. Addition of 10 mM MG132, a 26S proteosome inhibitor (Lee et al., 2009), blocked the low Pi–induced decrease of WRKY6 protein (Figures 7B and 7C), suggesting that a 26S proteasome–mediated WRKY6 proteolysis is involved in WRKY6-regulated PHO1 expression in response to low Pi stress. WRKY42 Interacts with the PHO1 Promoter and Negatively Regulates PHO1 Transcription Figure 3. ChIP Assays for At WRKY6 Binding to the W-Box of the PHO1 To identify other proteins that interact with WRKY6, we per- Promoter in Vivo. formed yeast two-hybrid experiments using WRKY6 as bait in (A) Diagram of the PHO1 promoter region showing the relative positions fusion with the Gal4 DNA binding domain. As listed in Supple- of four of six W-boxes (Q, 1718 to 1625; X, 1269 to 1181; Y, 966 mental Table 1 online, there are at least a dozen proteins that to 936; and Z, 775 to 618). W-boxes are marked by black rectan- interact with WRKY6. Among these WRKY6 interacting proteins, gles, and the untranslated region and exons of PHO1 are marked by gray WRKY42, as the closest homolog of WRKY6 (Eulgem et al., boxes. 2000), may have similar function to WRKY6. To test this hypoth- (B) ChIP-qPCR analysis of the PHO1 promoter sequence. ChIP assays were performed with chromatin prepared from wild-type Arabidopsis esis, we tested possible binding of WRKY42 with the PHO1 roots. The gray and black bars represent the ChIP signals with (WRKY6) promoter. The ChIP-qPCR experiments showed that, similar to and without (NoAB) addition of anti-WRKY6 serum, respectively. The WRKY6, WRKY42 can bind to both the Y and Z W-box motifs experiments were repeated three times, and three replicates were within the PHO1 promoter but not to the Q and X W-box motifs included for each sample in each experiment. The data are presented (Figure 8A). To further test possible function of WRKY42 on as means 6 SE (n =3). regulation of PHO1 expression, transient expression experi- ments in tobacco leaves were performed. The results showed wild-type roots was increased after the plants had been trans- that, similar to WRKY6, WRKY42 inhibited PHO1 promoter ferred to the low Pi medium for 3 d (Figure 5B; Ribot et al., 2008). activity (Figure 8B). The coinjection of Super:WRKY6 and Su- It was further proposed that the low Pi stress might trigger the per:WRKY42 showed much stronger repression on ProPHO1: plant responses through suppression of WRKY6 expression. GUS expression than did injection of either Super:WRKY6 or However, as shown in Figure 5A, after wild-type plants were Super:WRKY42 alone (Figure 8B). However, WRKY75 had no challenged with low Pi stress, WRKY6 expression level was effect on PHO1 expression and did not influence the inhibition of increased during the first 3 h and then decreased, but stayed PHO1 expression by WRKY6 (Figure 8B). above its zero time expression level for ;48 h. Another hypoth- Taking all these results together, we concluded that WRKY6 esis we proposed was that the Pi starvation inhibits WRKY6 functions in plant responses to low Pi stress by negatively functioning in suppression of PHO1 expression, such as through regulating PHO1 expression. Under normal conditions with suf- a possible blockage of WRKY6 binding to W-box motifs of the ficient Pi supply, WRKY6 (and probably also WRKY42) can bind PHO1 promoter or a low Pi–induced WRKY6 protein degrada- to the W-box motifs W and W within the PHO1 promoter and Y Z tion. ChIP-qPCR experiments were conducted to test whether represses the transcription of PHO1. Under Pi-deficient condi- WRKY6 protein still can bind to W and W boxes of the PHO1 Y Z tions, WRKY6 protein content is decreased via a 26S proteo- promoter under the low Pi condition. As shown in Figure 5C, the some–mediated proteolysis, and the interaction of WRKY6 and interaction between the WRKY6 protein and W or W box of the Y Z the PHO1 is limited. As a result, repression of PHO1 transcription PHO1 promoter was severely impaired under the low Pi condi- by WRKY6 is relieved, which might be important for plant tion. To confirm further the interaction of WRKY6 with the W and adaptation to a Pi-deficient environment. W boxes of the PHO1 promoter, ChIP-qPCR experiments were performed using the wild type, wrky6-1 mutant, and three WRKY6-overexpressing lines. As shown in Figure 6, the strong DISCUSSION interaction of WRKY6 with W or W boxes of PHO1 promoter Y Z was displayed again in all three WRKY6-overexpressing lines Plant-specific WRKY transcription factor family proteins have under the normal conditions (on MS medium), while this interac- been implicated in the regulation of genes involved in plant tion was reduced under the low Pi condition (on LP medium). responses to biotic as well as abiotic stresses, such as patho- It was further proposed that low Pi stress may induce degra- gen-induced stress (Dong et al., 2003; Eulgem and Somssich, dation of WRKY6 protein so that repression of PHO1 transcrip- 2007), drought, cold, and salinity stresses (Seki et al., 2002; Dong WRKY6 Regulates PHO1 Expression 3559 et al., 2003). WRKY factors act primarily by binding to conserved W-box elements in the promoters of specific targets to direct temporal and spatial expression of these genes (Ulker and Somssich, 2004). Among 74 members in the Arabidopsis WRKY family, only WRKY75 has been reported to be involved in modulation of Pi acquisition and root development (Devaiah et al., 2007a). This study demonstrated that WRKY6 (and prob- ably also WRKY42) plays an important role in modulation of plant responses to low Pi stress via regulation of PHO1 expression. WRKY6 Is a Negative Regulator for PHO1 Transcription Plant responses to Pi starvation involve the transcriptional reg- ulation of numerous genes to establish an adaptive mechanism (Franco-Zorilla et al., 2004). Several Arabidopsis transcription factors were identified functioning in the Pi starvation response, such as PHR1 (Rubio et al., 2001), ZAT6 (Devaiah et al., 2007b), BHLH32 (Chen et al., 2007), MYB62 (Devaiah et al., 2009), and a WRKY family protein WRKY75 (Devaiah et al., 2007a). WRKY75 can be induced by low Pi stress and is believed act as a positive regulator of Pi acquisition under Pi-deficient conditions (Devaiah et al., 2007a). In this study, we first observed that the WRKY6 overexpression lines displayed similar phenotypes as the pho1 mutant under low Pi stress, including growth inhibition and anthocyanin accumu- lation. We further demonstrated that WRKY6 protein can bind to two W-boxes of the PHO1 promoter and that PHO1 transcription was repressed by overexpression of WRKY6 under normal Pi supply conditions. This repression of PHO1 transcription by WRKY6 was relieved under low Pi conditions, indicating that the regulation of PHO1 transcription by WRKY6 is Pi dependent. In addition, protein blot analysis showed that the low Pi treatment– induced WRKY6 decrease was inhibited by a 26S proteosome inhibitor MG132. This suggests that the low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated WRKY6 proteolysis. Such a Pi-dependent mechanism may make WRKY6 a key regulator for plant responses to low Pi stress. Mechanism of PHO1 Regulation by WRKY6 and WRKY42 Under normal growth conditions, WRKY6 represses PHO1 ex- pression to balance Pi homeostasis through its binding to two W-boxes at the end of the coding region of the PHO1 promoter. When a low Pi stress signal is sensed by an unknown signaling mechanism and relayed to the E3 ubiquitin ligase, a 26S proteosome–mediated WRKY6 protein degradation is activated and WRKY6 binding to the PHO1 promoter W box motifs is weakened so that PHO1 transcription is induced to cope with the Figure 4. Suppression of PHO1 Expression by WRKY6. Pi-deficient environment. As a result, PHO1-facilitated Pi loading from root to xylem occurs and translocation of Pi from root to (A) RNA gel blot analysis of PHO1 expression in the roots of the WRKY6- overexpressing lines (Super:WRKY6-13, Super:WRKY6-18, and 35S: shoot could be promoted so that plants can adapt to a Pi- WRKY6-9), the wrky6-1 mutants, and wild-type plants. rRNA is shown as deficient environment. The plant may use WRKY6 (and probably a loading control. (B) to (D) GUS staining showing expression patterns of PHO1 in trans- genic plants carrying distinct PHO1 promoter constructs (indicated three independent transgenic lines for each background. All PHO1 above each panel; green boxes show W boxes, and yellow box repre- promoter–driven GUS transgenic lines are homozygous lines, and each sents the GUS gene) in 35S:WRKY6-9, wrky6-1 mutant, or wild-type line contains a single copy of insertion. backgrounds. The three roots in each group are representatives from (E) Relative GUS activities in different transgenic plants. 3560 The Plant Cell also WRKY42) as a key regulator that responds to varied Pi supply conditions and regulates Pi distribution in different organs via regulation of PHO1 as well as other unknown components. Our results also showed that, similar to WRKY6, WRKY42 can bind to both the Y and Z W-box motifs of the PHO1 promoter but not to the Q and X W-box motifs (Figure 8A). In addition, WRKY42 alone can also inhibit PHO1 expression (Figure 8B). Alternatively, considering that WRKY6 can interact with WRKY42 (see Sup- plemental Table 1 online), one may wonder if these two factors can form heterocomplexes to regulate PHO1 expression. Xu et al. (2006b) reported that three different kinds of WRKY proteins (WRKY18, WRKY40, and WRKY60) can interact with each other and form heterocomplexes, and the interactions between these WRKY factors influence their DNA binding activities. Robatzek and Somssich (2002) showed that WRKY6 can act as a negative regulator of its own and WRKY42 expression even though the mechanism and function remains unknown. To test possible synergic effects of WRKY6 and WRKY42 on PHO1 expression, we coinjected Super:WRKY6 and Super: WRKY42 in tobacco leaves to test their effects on ProPHO1: GUS expression. The results (Figure 8B) indicate that WRKY6 and WRKY42 together had stronger repression on PHO1 ex- pression. However, still we cannot conclude that they work together (by forming heterodimers) or work independently at this point. The ChIP-qPCR data showed that WRKY6 and WRKY42 had differential interactions with Y and Z W-boxes within the PHO1 promoter. WRKY6 displayed a stronger interaction with the Y box than with the Z-box (Figures 3B and 5C), while WRKY42 displayed a stronger interaction with the Z-box than with the Y-box (Figure 8A). These results indicate that WRKY6 and WRKY42 may regulate PHO1 expression in different ways. To clarify further if WRKY6 and WRKY42 work independently or together as a complex in regulation of PHO1 expression is an important issue for comprehensively understanding complex mechanisms of PHO1 regulation by WRKY factors. Regulation of PHO1 by Other Possible Regulatory Factors Although the Q and X W-boxes within the PHO1 promoter do not bind to WRKY6, we have noticed that the deletion of the sequences containing the Q- and X-boxes reduced the inhibitory effect of WRKY6 on PHO1 expression (Figures 4B, 4C, and 4E), particularly for wild-type plants. One of possible explanation for Figure 5. Repression of PHO1 Expression by WRKY6 Was Released in this phenomenon is that, within the deleted sequences, some Response to Low Pi Stress. other regulatory elements related to plant responses to low Pi stress may exist. The analysis of the deleted sequences using (A) qPCR analysis of WRKY6 expression induced by Pi starvation. TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess) shows that, (B) qPCR analysis of PHO1 expression induced by Pi starvation. (C) ChIP-qPCR assays to detect the association between WRKY6 and in addition to Q and X W-boxes, there are a number of regulatory W-boxes within the PHO1 promoter in wild-type plants under the normal elements for the following transcription factors: GATA factors, (MS) and LP conditions. The ChIP signals with (WRKY6) and without CCAAT-box transcription factor, multiprotein bridging factor (NoAB) addition of anti-WRKY6 serum are indicated. The data are 1 (MBF1), homeodomain-leucine zipper (HD-Zip) transcription presented as means 6 SE (n = 3). The experiments were repeated three factor, MYB transcription factor, Dof (DNA binding with one times, and three replicates were included for each sample in each finger) factor, heat shock transcriptional factor (HSF), etc. Al- experiment. though there is no report so far regarding PHO1 regulation by these TFs, at least two of them have been reported involving plant responses to low Pi stress. The HD-Zip factor has been reported to bind to the phosphate response domain of the soybean (Glycine max) VspB tripartite promoter (Tang et al., WRKY6 Regulates PHO1 Expression 3561 Figure 6. ChIP-qPCR Assays to Detect the Association of WRKY6 and the PHO1 Promoter in the Tested Plants as Indicated under Pi-Sufficient (MS) and Pi-Deficient (LP) Conditions. ChIP assays were performed with chromatin prepared from tested plants roots to analyze the binding of At WRKY6 protein to the W -box ([A]; Q site), W -box ([B]; X site), W -box ([C]; Y site), and W -box ([D]; Z site) of the PHO1 promoter in vivo. The ChIP signals with (WRKY6) and without (NoAB) X Y Z addition of anti-WRKY6 serum are indicated. The experiments were repeated three times, and three replicates were included for each sample in one experiment. The data are presented as means 6 SE (n = 3). 2001). Nilsson et al. (2007) reported that increased expression of et al., 1991; Hamburger et al., 2002). Considering these results the MYB-related transcription factor PHR1 resulted in enhance- together with the fact that WRKY75 did not have an effect on ment in phosphate uptake in Arabidopsis. It is plausible to further PHO1 promoter activity (Figure 8B; in addition, WRKY75 did not hypothesize that one or more of these regulatory elements (which interact with WRKY6 in the yeast two-hybrid assay), we may were deleted together with Q and X W-boxes in the experiments further hypothesize that WRKY75 and WRKY6, in response to shown in Figure 4C) may directly or indirectly be involved in low Pi stress, function in different regulatory pathways. Identify- PHO1 regulation. ing the gene(s) whose expression is specifically regulated by WRKY75 as well as other possible transcription factors will help us to clarify the complex mechanisms of plant responses to low WRKY6 and WRKY75 May Respond to Low Pi Stress via Pi stress. Different Pathways WRKY75 has been reported to play an important role in the Other Possible Roles of WRKY in Regulation of Pi Starvation phosphate starvation response, particularly by modulating Pi Responsive Genes uptake and root development (Devaiah et al., 2007a). The results presented here demonstrate that WRKY6 responds to low Pi To test if WRKY6 would play roles in regulation of other Pi stress by regulation of PHO1 transcription. Repression of starvation responsive genes, we performed comparative tran- WRKY75 expression (by RNA interference methods) resulted in scriptome analyses with various plant materials (35S:WRKY6-9, a decrease of Pi uptake (Devaiah et al., 2007a), while over- wrky6-1, and the wild type) using the Affymetrix GeneChip. As expression of WRKY6 repressed PHO1 expression and conse- shown in Supplemental Table 2 online, among 30 low Pi re- quently reduced Pi accumulation in shoots. It is known that sponse genes (Devaiah et al., 2007a; Lin et al., 2009), 11 of them PHO1 functions in Pi translocation from root to shoot (Poirier showed expression changes between either wild-type and 35S: 3562 The Plant Cell In addition, among the genes whose expression was upregu- lated or downregulated by more than two times and showed relevant changes (changes in opposite direction in 35S:WRKY6-9 plants compared with the wrky6-1 mutant), there are a total of 25 genes (listed in Supplemental Table 3 online) whose pro- moters contain W-box(es). Among these genes, there are 15 genes whose transcriptions were repressed, and transcription of 10 other genes was enhanced in 35S:WRKY6-9 plants. These data indicate that, in addition to its function in plant responses to low Pi stress, WRKY6 may be involved in a broad range of transcriptional regulations related to different processes, such as senescence, pathogen defense, and wounding responses (Robatzek and Somssich, 2001, 2002). Figure 7. WRKY6 Protein Blot Analysis. Seven-day-old wild-type seedlings were transferred to LP medium (A), LP medium with 10 mM MG132 (LP+MG132) (B), or LP medium with DMSO (LP+DMSO) (C). The roots of seedlings were harvested for protein extraction at the indicated time. Protein extracts were analyzed by immunoblots using rabbit anti-WRKY6 serum. Tubulin levels were detected in parallel as a loading control with antitubulin antibody. WRKY6-9 plants or the wild type and the wrky6-1 mutant. Among the members of the PHT1 family, Pht1;5 and Pht1;8 displayed transcriptional changes in 35S:WRKY6-9 plants and the wrky6-1 mutant compared with wild-type plants (see Supplemental Table 2 online). Both Pht1;5 and Pht1;8 contain W-boxes in their promoters (Devaiah et al., 2007), suggesting that WRKY6 may regulate their transcription. It is known that expression of Pht1;8 was significantly increased in the pho2 mutant (Aung et al., 2006; Bari et al., 2006), a mutant overaccumulating Pi in leaves, suggesting a possible role of Pht1;8 in WRKY6- and PHO1- Figure 8. Suppression of PHO1 Expression by WRKY42. related Pi mobilization. PS2 and PS3, two members of a phos- (A) ChIP-qPCR assays to detect the association between WRKY42 and phatase family, were significantly upregulated in 35S:WRKY6-9 W-boxes within the PHO1 promoter in wild-type plants under normal plants and downregulated in the wrky6-1 mutant (see Supple- conditions. The experiments were repeated three times, and three mental Table 2 online), suggesting that WRKY6 also might be replicates were included for each sample in each experiment. The data involved in plant early responses to low Pi stress (Devaiah et al., are presented as means 6 SE (n = 3). 2007). Several low Pi responsive transcription factors listed in (B) Transient overexpression of the ProPHO1:GUS fusion together with Supplemental Table 2 online, including PHR1, ZAT6, WRKY75, Super:WRKY6, Super:WRKY42,or Super:WRKY75 in Nicotiana ben- and BHLH32, did not show significant changes in their tran- thamiana leaves. ProPHO1:GUS fusion together with Super1300 vector scription either in 35S:WRKY6-9 plants or in the wrky6-1 mutant. was taken as the control. The data are presented as means 6 SE (n = 4). WRKY6 Regulates PHO1 Expression 3563 METHODS GATTTCAT-39 and the reverse primer 59-TTGGCGGCACCCTTACGTG- GATCA-39). EF1a was used as a quantitative control. For real-time PCR analysis, total RNA extraction was performed as Plant Materials and Growth Conditions described above, and the RNA was treated with DNase I RNase Free The WRKY6 overexpression lines 35S:WRKY6-3, 35S:WRKY6-5, 35S: (Takara) to eliminate genomic DNA contamination. The cDNA was syn- WRKY6-9, and the WRKY6 knockout mutant wrky6-1 were kindly pro- thesized from total RNA by SuperScript II RNase H reverse transcriptase vided by Imre E. Somssich (Max-Planck-Institut, Germany; Robatzek and (Invitrogen) using Radom Hexamer Primer (Promega). Quantitative real- Somssich, 2002). The Super:WRKY6-13 and Super:WRKY6-18 lines were time PCR was performed using the Power SYBR Green PCR Master Mix generated by cloning the coding sequence of WRKY6 into Super1300 (Applied Biosystems; P/N 4368577) on a 7500 Real Time PCR System vector (Li et al. 2001). The pho1 mutant was ordered from the ABRC machine (Applied Biosystems) following the manufacturer’s protocols. (http://www.Arabidopsis.org/abrc/). The PCR amplification was performed at 958C for 15 s and 608C for 1 min. For phenotype tests and seed harvest, Arabidopsis thaliana plants were Relative quantitative results were calculated by normalization to 18S grown in a potting soil mixture (rich soils:vermiculite = 2:1, v/v) and kept in rRNA. qPCR was conducted with WRKY6-specific primers (the forward 22 21 growth chambers at 228C with illumination at 120 mmol·m ·s for an primer 59-TAGTCACGACGGGATGATGA-39 and the reverse primer 18-h daily light period. The relative humidity was ;70% (65%). 59-ATTAGGAGGCGGAGGTGAGT-39)and PHO1-specific primers (the Low Pi stress treatment of plants was conducted by growing seedlings forward primer 59-TGGTTCTCCGGAACAAGAAC-39 and the reverse on Petri dishes containing Pi-sufficient (MS) or Pi-deficient (low Pi or LP) primer 59-TGACTTCAAGTGACGCCAAG-39). medium. The seeds were surface sterilized with the mixed solutions of NaClO (0.5%) and Triton X-100 (0.01%) for 10 min followed by washing Antibody Generation and ChIP-qPCR Assay with sterilized distilled water four times. The sterilized seeds were first The whole coding sequences were amplified by PCR using WRKY6- incubated on Petri dishes containing MS agar (0.8%) medium (containing specific primers (the forward primer 59-cccgggCCCGGGATGGACAGAG- 1.25 mM KH PO and 3% sucrose) at 48C for 2 d before germination. The 2 4 GAGGTCT-39 and the reverse primer 59-ctcgagCTCGAGCTATTGATTT- seeds were germinated at 228C under constant illumination at 40 22 21 TTGTTGTTTC-39)and WRKY42-specific primers (the forward primer mmol·m ·s and the 7-d-old seedlings were transferred to LP medium. 59-ggatccGGATCCATGTTTCGTTTTCCGGTAAG-39 and the reverse primer The LP medium was made by modification of MS medium such that the Pi 59-gagctcGTCGACTCTTATTGCCTATTGTCAAC-39). The lowercase let- (supplied with KH PO ) concentration in LP medium was 10 mM, and agar 2 4 ters represent the restriction sites. The whole coding sequences of At was replaced by agarose (Promega) to avoid the contamination of WRKY6 and WRKY42 were cloned into pGEX-4T-2 (Pharmacia). The phosphorous. reconstructed pGEX-4T-2 plasmid containing WRKY6 or WRKY42 was then transformed into Escherichia coli strain BL21 (DE3) to express Quantification of Total Pi proteins by induction with 0.1 mM isopropyl-b-D-thiogalactopyranoside Seven-day-old Arabidopsis seedlings germinated on MS medium were at 188C for 8 h. The resulting glutathione S-transferase (GST) fusion transferred to MS or LP medium for another 7 d, and then the roots and proteins were purified using Glutathione Sepharose 4B (Pharmacia). shoots were harvested for Pi content measurements. The samples were Protein concentrations were determined using the Bio-Rad protein assay oven dried at 808C for 48 h before determination of dry weight, and the kit. The polyclonal antibodies against WRKY6 or WRKY42, generated by samples were ashed in a muffle furnace at 3008C for 1 h and 5758Cfor an inoculation of rabbits, were purified using the Amino Link Plus Immobi- additional 5 h and then dissolved in 0.1 N HCl. The total Pi content in the lization Kit (Pierce). samples was quantified as described previously (Ames, 1966). The 7-d-old seedlings germinated on MS medium were transferred to MS or LP medium for another 7 d and then the roots were harvested for ChIP experiments. The chromatin samples for ChIP experiments were RNA Gel Blot, RT-PCR, and Real-Time PCR Analysis obtained following the methods by Saleh et al. (2008). The roots of plants For RNA gel blot analysis, 7-d-old Arabidopsis seedlings germinated on seedlings were first cross-linked by formaldehyde, and the purified cross- MS medium were transferred to MS or LP medium for another 7 d and linked nuclei were then sonicated to shear the chromatin into suitably then the seedlings or roots were harvested for extraction of total RNA sized fragments. The antibody that specifically recognizes the recombi- using the Trizol reagent (Invitrogen). Thirty micrograms of RNA were nant WRKY6-GST or WRKY42-GST was used to immunoprecipitate loaded per lane and transferred to a nylon membrane for hybridization. DNA/protein complexes from the chromatin preparation. The DNA in the Gene-specific probes were amplified by PCR using WRKY6-specific precipitated complexes was recovered and analyzed by qPCR methods. primers (the forward primer 59-CTTTGGCGATGTCTAGAATTGA-39 and Following the methods described by Haring et al. (2007), qPCR analysis the reverse primer 59-CCTCACCTACTGCTCTCGTAGG-39) and PHO1- was performed using the Power SYBR Green PCR Master Mix (Applied specific primers (the forward primer 59-TACTTGATTCTTTCTTACCC- Biosystems; P/N 4368577) in a 20-mL qPCR reaction on the 7500 Real TACTTCTGG-39 and the reverse primer 59-TCCAAGGAACGGTAACGG- Time PCR System machine (Applied Biosystems) following the manu- TACGGTCTTCACT-39) as the templates, respectively. The probes were facturer’s protocols. The chosen primer combinations (see Supplemental labeled with [a- P]dCTP using random primer labeling reagents (Phar- Table 4 online) can amplify fragments of 150 to 200 bp within the promoter macia) and hybridized to RNA gel blotted onto nylon membrane. The of PHO1. To ensure the reliability of ChIP data, the input sample and no- rRNA was taken as the control. antibody (NoAB) control sample were analyzed with each primer set. The For RT-PCR analysis, total RNA was extracted with Trizol reagent results were quantified with a calibration line made with DNA isolated (Invitrogen) and then treated with DNase I RNase Free (Takara) to from cross-linked and sonicated chromatin. eliminate genomic DNA contamination. The cDNA was synthesized from treated RNA by SuperScript II RNase H reverse transcriptase Vector Construction and Arabidopsis Transformation and (Invitrogen) using oligo(dT) primer (Promega). The PCR experiments GUS Assays were conducted with WRKY6-specific primers (the forward primer 59-ATGTTTCGTTTTCCGGTAAGTCTTGGAGGA-39 and the reverse PHO1 promoter variants, including the full-length promoter (2282 bp), the primer 59-TATTGCCTATTGTCAACGTTGCTCGTTGTAACATTA-39)and promoter containing two W-boxes (Y and Z, 1141 bp), and the promoter EF1a -specific primers (the forward primer 59-ATGCCCCAGGACATCGT- without W-box (454 bp), were amplified by PCR from Arabidopsis 3564 The Plant Cell genomic DNA and cloned into the transformation vector pCAMBIA1381 1.0 (MAS 5.0) software to generate detection calls and normalized using at the SalIand PstI restriction sites, respectively. All primer sequences Affymetrix GCOS software, and the TCT value was set to 100. When used for vector constructions are listed in Supplemental Table 5 online. All analyzing the transcriptionally changed genes, the signal ratio between tested Arabidopsis plants (Col-0, 35S:WRKY6-9, and the wrky6-1 mutant) two plant materials was calculated to represent the fold change of this were transformed by these three constructed vectors, respectively, using gene for its transcription in the corresponding process, and the fold the Agrobacterium tumefaciens–mediated gene-transfer procedure change P value for each gene was obtained at the same time using GCOS (Clough and Bent, 1998). All transgenic lines used in this study are T3 software. A gene was considered to be transcriptionally changed when it homozygous plants with single copy insertion. The GUS staining and GUS was upregulated or downregulated more than onefold. The genes with P activity measurements were performed as described previously (Xu et al., values < 0.05 and fold changes > 1 are included in Supplemental Table 3 2006a). online. Accession Numbers SDS-PAGE and Protein Gel Blot Analysis Sequence data from this article can be found in the Arabidopsis Genome For MG132 treatment, 10 mM stock solution was made by dissolving Initiative or GeneBank/EMBL database under the following accession MG132 (Calbiochem) in DMSO, and the final MG132 concentration was numbers: WRKY6 (At1g62300), WRKY42 (At4g04450), WRKY75 10 mM. For the control of MG132 treatment, 1/1000 DMSO was added to (At5g13080), PHO1 (At3g23430), and EF1a (At5g60390). Microarray the medium. The 7-d-old seedlings germinated on MS medium were data from this article have been deposited with the National Center for transferred to low Pi (LP), low Pi with 10 mM MG132 (Calbiochem) Biotechnology Information Gene Expression Omnibus data repository (LP+MG132), or low Pi with DMSO (LP+DMSO) medium, and then the (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18273. roots were harvested at the indicated time for immunoblot analysis. Total proteins were extracted according to the method of Saleh et al. (2008), Supplemental Data and 50 mg proteins of each sample were separated on a 10% SDS-PAGE and analyzed by protein gel blot according to Towbin et al. (1979). Mouse The following materials are available in the online version of this article. anti-tubulin (Sigma-Aldrich) (with 1:4000 dilution) and rabbit anti-WRKY6 Supplemental Figure 1. Phenotype Test of WRKY6-Overexpressing serum (with 1:10,000 dilution) were used as the primary antibodies, and Lines. goat anti-mouse (or anti-rabbit) peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson) (with 1:8000 dilution) was used as the Supplemental Figure 2. Pi Content Measurements in Various Plant secondary antibody. The membranes were visualized using a Super- Materials. Signal West Femto Trial Kit (Themor) following the manufacturer’s in- Supplemental Figure 3. Phenotype Comparison of the wrky6-1 Mu- structions. tant, 35S:WRKY6-9, and Wild-Type Plants under a Short-Day (10-h- Light/14-h-Dark) Condition. Transient Expression Assays in Nicotiana benthamiana Supplemental Table 1. Yeast Two-Hybrid Results with At WRKY6 as the Bait. The constructs of ProPHO1:GUS with Super1300, Super1300:WRKY6,or Super1300:WRKY42 were transformed into A. Agrobacterium (GV3101). Supplemental Table 2. Expression of Pi Starvation Responsive Agrobacterium cells were harvested by centrifugation and suspended in Genes in the 35S:WRKY6-9 Plant and the wrky6-1 Mutant. the solutions containing 10 mM MES, pH 5.6, 10 mM MgCl , and 200 mM Supplemental Table 3. Genes with W-Boxes Whose Expression Was acetosyringone to an optical density (600 nm) of 0.7, incubated at room Up- or Downregulated More Than Twofold in the 35S:WRKY6-9 Line temperature for 4 h, and then used to infiltrate leaves of N. benthamiana (OE) and the wrky6-1 Mutant (KO) Compared with Wild-Type Plants. using a needle-free syringe. The GUS activity of the infiltrated leaves was quantitatively determined. Supplemental Table 4. Primers Used for ChIP-qPCR Experiments. Supplemental Table 5. Primers Used for the ProPHO1:GUS Con- Yeast Two-Hybrid Assay structs. The WRKY6 full-length cDNA sequence was amplified with WRKY6- specific primers (the forward primer 59-GCcatatgATGGACAGAG- ACKNOWLEDGMENTS GATGGTCTGG-39 and the reverse primer 59-GCgtcgacCTATT- This work was supported by a competitive research grant (30421002) for GATTTTTGTTGTTTCCTTCG-39) and cloned into pGBK-T7 vector at Creative Research Groups sponsored by the National Science Founda- NdeI and SalI sites. The lowercase letters in the primers indicate restric- tion of China and the Programme of Introducing Talents of Discipline to tion sites. Two-hybrid screening and assays were performed as de- Universities (no. B06003). We thank Imre E. Somssich (Max-Planck- scribed (Kohalmi et al., 1997). Strain AH109 (BD Biosciences) of Institut fu¨rZu¨ chtungsforschung, Abteilung Biochemie, Germany) for Saccharomyces cerevisiae was used in all yeast two-hybrid experiments. kindly providing At WRKY6 overexpression lines 35S:WRKY6-3, 35S: WRKY6-5, and 35S:WRKY6-9 and the At WRKY6 knockout mutant Microarray Analysis (wrky6-1). 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The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in Arabidopsis  

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Oxford University Press
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© 2009 American Society of Plant Biologists
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1040-4651
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1532-298X
DOI
10.1105/tpc.108.064980
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Abstract

The Plant Cell, Vol. 21: 3554–3566, November 2009, www.plantcell.org ã 2009 American Society of Plant Biologists The WRKY6 Transcription Factor Modulates PHOSPHATE1 W OA Expression in Response to Low Pi Stress in Arabidopsis 1 1 1 2 Yi-Fang Chen, Li-Qin Li, Qian Xu, You-Han Kong, Hui Wang, and Wei-Hua Wu State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, National Plant Gene Research Centre (Beijing), Beijing 100193, China Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and b-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression. INTRODUCTION PHO1 is predominantly expressed in the stellar cells of the root and the lower part of the hypocotyls and is believed have a role in Phosphorus (P), as a major essential nutrient for plant growth and Pi efflux out of root stellar cells for xylem loading (Hamburger development, serves various basic biological functions in the et al., 2002). However, PHO1 shares no homology with any plant life cycle (Raghothama, 1999). Phosphate (H PO ,orin 2 4 previously described Pi transporter proteins in plants and fungi short, Pi) is the major form that is most readily taken up and (Hamburger et al., 2002). It is interesting that PHO1 contains a transported in the plant cell (Ullrich-Eberius et al., 1981; Tu et al., SPX domain, which can be found in several proteins that are 1990). The Pi concentration in the soil, typically 10 mM or less, involved in phosphate transport and/or Pi signaling pathways in results in Pi starvation for plant growth and survival, which is one plants and yeast. For example, an SPX protein in yeast named of major limiting factors for crop production in the cultivated PHO81 is a key regulator in transporting and sensing phosphate, soils. A number of studies have shown that plants have evolved as well as in sorting proteins to endomembranes (Lenburg and different strategies to overcome limited Pi availability. In re- O’Shea, 1996; Wykoff and O’Shea, 2001). In Arabidopsis, the sponse to low Pi stress or Pi starvation, plants may increase the SPX proteins SPX1-SPX3 are involved in Pi signaling pathways Pi uptake from the soil by alteration of root architecture and and regulate the expression of the Pi transporter genes Pht1;4 function (Lo´ pez-Bucio et al., 2003; Ticconi and Abel, 2004; and Pht1;5 (Duan et al., 2008). Thus, the possibility cannot be Osmont et al., 2007). Under Pi-limiting conditions, plants may excluded that PHO1 may not be a direct Pi transporter but rather also increase their Pi acquisition by changing their metabolic and may regulate Pi loading of the xylem either by directly influencing developmental processes (Raghothama and Karthikeyan, 2005), the activity of transporter proteins or via signal transduction. such as increasing phosphatase activity (Lipton et al., 1987) and PHO1 gene expression can be induced by Pi starvation secretion of organic acids (Marschner, 1995). (Stefanovic et al., 2007; Ribot et al., 2008; also see Figure 5B in PHOSPHATE1 (PHO1) has been shown to play roles in Pi this study), but the transcription factors that regulate PHO1 translocation from root to shoot (Hamburger et al., 2002), which expression remain unknown. Transcriptome analysis has dem- is also important for plant adaptation to a low Pi environment. A onstrated that expression of many genes is significantly changed single nuclear recessive mutation in PHO1 led to its inability to in Oryza sativa (Wasaki et al., 2003) and Arabidopsis thaliana (Wu load Pi into xylem (Poirier et al., 1991; Hamburger et al., 2002). et al., 2003; Misson et al., 2005) under Pi-limiting conditions, indicating that transcriptional regulation may play important roles These authors contributed equally to this work. in plant responses to low Pi stress. More recently, a number of Address correspondence to wuwh@public3.bta.net.cn. regulatory components that may be involved in plant responses The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described to low Pi stress have been reported, such as microRNA miR339 in the Instructions for Authors (www.plantphysiol.org) is: Wei-Hua Wu (Bari et al., 2006; Chiou et al., 2006), Arabidopsis posttranslation (wuwh@public3.bta.net.cn). regulators PHOSPHATE TRANSPORTER TRAFFIC FACILITA- Online version contains Web-only data. OA TOR1 (At PHF1) (Gonza´ lez et al., 2005) and E3 SUMO Ligase Open Access articles can be viewed online without a subscription. www.plantcell.org/cgi/doi/10.1105/tpc.108.064980 (At SIZ1) (Miura et al., 2005), transcription factors PHOSPHATE WRKY6 Regulates PHO1 Expression 3555 STARVATION RESPONSE1 (At PHR1) (Rubio et al., 2001) and O. plants were grown in a potting soil mixture (Figure 1A). However, sativa Pi STARVATION-INDUCED TRANSCRIPTION FACTOR1 the overall growth of the WRKY6 null mutant wrky6-1,an En-1 (Os PTF1) (Yi et al., 2005), and Arabidopsis MYB62 transcription insertion mutant, was obviously better than wild-type plants factor (At MYB62) (Devaiah et al., 2009) and Arabidopsis (Figure 1A). The measured free Pi concentration in the potting WRKY75 transcription factor (At WRKY75) (Devaiah et al., soils was ;10 mM in this study. Thus, the plants grown under the 2007a). described conditions as shown in Figure 1A were actually WRKY proteins are plant-specific transcription factors encoded experienced low Pi stress. Under this low Pi stress condition, by a multigene family comprising 74 members in Arabidopsis the 35S:WRKY6-9 line displayed thinner stalks and smaller (http://www.Arabidopsis.org/browse/genefamily/WRKY-Som. leaves compared with wild-type plants (Figure 1A). Figure 1B jsp), and many of them have been found to play important roles in shows that the En-1 insertion in wrky6-1 disrupted the transcrip- plant responses to biotic and abiotic stresses. In addition to a tion of the WRKY6 gene. number of WRKY genes that have been demonstrated to be Besides the 35S:WRKY6-9 line, two more WRKY6-overex- involved in plant responses to pathogen infection and other pressing lines, Super:WRKY6-13 and Super:WRKY6-18, were defense-related stimuli (Dong et al., 2003; Kalde et al., 2003; Li included in our further experiments. The elevated expression of et al., 2004; Eulgem and Somssich, 2007), some WRKY genes WRKY6 mRNA in these transgenic lines is shown in Figure 1C. have also been shown to function in plant responses to various Transcription of WRKY6 in either wild-type or the wrky6-1 plants abiotic stress, such as drought (Pnueli et al., 2002; Rizhsky et al., was not detectable in our RNA gel blot experiments. Among the 2002; Seki et al., 2002; Mare, et al., 2004), cold (Huang and three WRKY6-overexpressing lines, 35S:WRKY6-9, Super: Duman, 2002; Seki et al., 2002; Mare, et al., 2004), heat (Rizhsky WRKY6-18, and Super:WRKY6-13 displayed the highest, me- et al., 2002), salinity (Seki et al., 2002), wounding (Hara et al., dium, and the lowest WRKY6 expression, respectively. 2000), and Pi starvation (Devaiah et al., 2007a). However, little is Plants usually accumulate more anthocyanin in their aerial known about the specific interaction of a given WRKY protein portions in response to low Pi stress (Marschner, 1995), and this with a defined target gene. Recent studies using an oligodeoxy- can result in brown-colored leaves. When grown on Murashige nucleotide decoy strategy have revealed that SUSIBA, a WRKY and Skoog (MS) medium with sufficient Pi supply, all tested protein, can bind to SURE (sugar responsive) and W-box ele- plants showed no difference in their phenotypes (top panel in ments in the iso1 promoter (Sun et al., 2003). Petroselinum Figure 1D). After the low Pi treatment, the WRKY6-overexpress- crispum WRKY1 has been shown to bind to the W-box of its ing plants, particularly Super:WRKY6-18 and 35S:WRKY6-9 native promoter as well as to that of Pc WRKY3 and Pc PR1-1 lines (both have much higher WRKY6 expression than does the based on chromatin immunoprecipitation (ChIP) analysis Super:WRKY6-13 line), displayed dark-brown leaves similar to (Turck et al., 2004). Candidate target genes for At WRKY53 the phenotype of the pho1 mutant (bottom panel in Figure 1D). To were identified with a pull-down assay (Miao et al., 2004), and confirm further the effects of WRKY6 overexpression on the low electrophoretic mobility shift assays identified candidate targets Pi response phenotype, another group of WRKY6-overexpress- for Hordeum vulgare WRKY transcription factor WRKY38 (Zou ing lines (35S:WRKY6-3, 35S:WRKY6-5, and 35S:WRKY6-9; et al., 2008), Arabidopsis WRKY26 and WRKY11 (Ciolkowski Robatzek and Somssich, 2002) were tested. As shown in Sup- et al., 2008), Nicotiana tabacum WRKY1, WRKY2, and WRKY4 plemental Figure 1 online, overexpression of WRKY6 indeed (Yamamoto et al., 2004), and O. sativa WRKY71 (Zhang et al., increased plant sensitivity to low Pi stress. Furthermore, increase 2004). of transgenic plant sensitivity to low Pi stress was closely related At WRKY6 was first reported to be associated with senes- to WRKY6 expression level (see Supplemental Figure 1 online). cence- and defense-related processes, and it could activate the A defect in Pi transfer from root to shoot has been reported in expression of its target gene SIRK, a receptor-like protein kinase the pho1 mutant (Poirier et al., 1991; Hamburger et al., 2002), in the process of senescence (Robatzek and Somssich, 2002). resulting in reduced Pi content in the shoot and smaller plant size. Here, we report a previously unknown function of WRKY6 in plant Under either Pi-sufficient or Pi-deficient conditions, the WRKY6- responses to low Pi stress. We demonstrate that plants over- overexpressing lines showed similar reduced Pi contents in expressing WRKY6 become more sensitive to low Pi stress and shoots as the pho1 mutant (Figures 2A and 2B). As a result, the display a similar phenotype as the pho1 mutant. WRKY6 nega- ratios of Pi content in shoot to that in root (Pi /Pi ) for both shoot root tively regulates PHO1 expression by binding to two W-box WRKY6-overexpressing lines and the pho1 mutant were signif- consensus motifs within the PHO1 promoter, and the repression icantly lower than the ratio determined in wild-type plants, of PHO1 expression by WRKY6 is released under low Pi condi- particularly under low Pi condition (Figures 2C and 2D). In tions. addition, four Super:PHO1 lines (Super:PHO1-1, -7, -9, and -13) with differential PHO1 expression were selected for the Pi content assay. As shown in Supplemental Figure 2 online, all Super:PHO1 lines and the wrky6-1 mutant displayed higher Pi RESULTS contents in shoots, whereas the pho1 mutant showed the lowest Pi content in shoots under both MS and low Pi (LP) conditions. WRKY6 Overexpression Plants Showed Similar Phenotypes The results demonstrate that Pi content in shoots indeed corre- as the pho1 Mutant under Low Pi Conditions lates with PHO1 expression. These data suggest that WRKY6 The growth of the aerial portion of the Arabidopsis WRKY6- may play a role in plant responses to Pi starvation at least partially overexpressing line (35S:WRKY6-9) was impaired when the through regulating PHO1-dependent Pi transfer. 3556 The Plant Cell It should be noted that the wrky6-1 mutants were obviously growing better than wild-type plants under the low Pi condition (Figures 1A and 1D, bottom panel) in our experiments, although they showed no difference under Pi-sufficient conditions (Figure 1D, top panel). However, Robatzek and Somssich (2001, 2002) had not ob- served phenotype difference between the wrky6-1 mutants and wild-type plants. After we have grown the plants under different environmental conditions, we believe that this difference mainly resulted from growth conditions, particularly light period. In the studies by Robatzek and Somssich (2001, 2002), plants were grown first under short-day conditions followed by long-day periods, while plants were grown under a constant long-day (18 h light) condition in our experiments. When Arabidopsis plants were grown under a short-day condition (10-h light), almost no phenotype difference between wild-type and wrky6-1 plants was observed (as shown in Supplemental Figure 3 online). WRKY6 Interacts with Two W-Box Motifs of the PHO1 Promoter To test the hypothesis that WRKY6 regulates PHO1 expression, we first tested whether WRKY6 could bind the PHO1 promoter. It is known that WRKY proteins usually bind to the W-box motifs of their target gene promoters (Eulgem et al., 2000). Analysis of the primary sequence of the PHO1 promoter revealed six W-box consensus motifs within the PHO1 promoter and four of them (named W ,W ,W , and W , respectively) are located at the very Q X Y Z end of promoter nearing the coding region (Figure 3A). The in vivo interaction between WRKY6 and the W-box motifs of the PHO1 promoter was investigated using the ChIP-qPCR (chromatin immunoprecipitation quantitative PCR) method. As shown in Figure 3B, WRKY6 strongly interacted with the PHO1 promoter when the primer combinations encompassing either W or W Y Z were applied, while no interaction was observed between WRKY6 and PHO1 promoter containing only W or W box. Q X These results demonstrated that WRKY6, as a transcription factor, can bind to two (W and W ) W-box motifs within the Y Z PHO1 promoter nearing the coding region, suggesting regulation of PHO1 transcription by WRKY6. WRKY6 Negatively Regulates PHO1 Transcription Based on the results of the phenotype tests (Figure 1), Pi content measurements (Figure 2), and ChIP analysis (Figure 3B), we further hypothesized that WRKY6 may negatively regulate PHO1 type seedlings. Seven-day-old seedlings were used for RNA extraction. EF1a was amplified for the control. Figure 1. Phenotype Tests of Various Plant Materials. (C) RNA gel blot analysis of WRKY6 expression in the WRKY6-over- (A) Phenotype comparison of the WRKY6-overexpressing line (35S: expressing lines (Super:WRKY6-13, Super:WRKY6-18,and 35S: WRKY6-9), the WRKY6 En-1 insertion mutant (wrky6-1), the pho1 mu- WRKY6-9) and the wrky6-1 mutant. Seven-day-old seedlings were tant, and wild-type (Columbia-0 [Col-0]) plants. All plants were grown in a used for RNAs extracted. The ethidium bromide–stained rRNA band potting soil mixture (rich soil:vermiculite = 2:1, v/v) and kept in growth was shown for the loading controls. 2 1 chambers at 228C with illumination at 120 mmol·m ·s for an 18-h daily (D) Phenotype comparison of the various plant lines as indicated. The light period for 30 d. 7-d-old seedlings germinated on MS medium were transferred to MS (B) RT-PCR test of WRKY6 expression in the wrky6-1 mutant and wild- (top panel) or LP (bottom panel) medium for another 7 d. WRKY6 Regulates PHO1 Expression 3557 Figure 2. Pi Content Measurements in Various Plant Materials. The 7-d-old seedlings of WRKY6-overexpressing lines (Super:WRKY6-13, Super:WRKY6-18, and 35S:WRKY6-9), the wrky6-1 mutant, the pho1 mutant, and wild-type plants germinated on MS medium were transferred to MS ([A] and [C])orLP([B] and [D]) medium for another 7 d, and then the shoots and roots of the seedlings were harvested separately for Pi content measurements. (A) and (B) Pi contents in roots and shoots of tested plant materials. Three replicates were included for each treatment, and experiments were repeated three times. Data are shown as means 6 SE (n =3). (C) and (D) Comparison of the ratio of Pi to Pi . The ratio was calculated from the data presented in (A) and (B).Dataare shownasmeans 6 SE (n =3). shoot root transcription. To test this hypothesis, we first compared the cating the removal of negative regulation (Figures 4B to 4E). On transcription of PHO1 in the roots of WRKY6-overexpressing the other hand, strong GUS expression was detected in wrky6-1 lines, wrky6-1, and wild-type plants, since both WRKY6 and roots regardless of which promoter fragment was used (Figures PHO1 are highly expressed in roots (Robatzek and Somssich, 4B to 4E). The GUS expression level was much higher in wrky6-1 2001; Hamburger et al., 2002). As shown in Figure 4A, the roots than in wild-type roots when the reporter gene was driven transcription of PHO1 in roots was repressed in the WRKY6- by promoters containing W and W (Figures 4B, 4C, and 4E). Y Z overexpressing lines. Repression of PHO1 expression was also However, there was almost no difference in the GUS staining closely related to the WRKY6 expression levels in WRKY6- among the roots of all three different types of plants when no overexpressing lines, with the strongest repression in 35S: W-box motif existed (Figures 4D and 4E). More importantly, in the WRKY6-9 plants and the weakest repression in Super: roots of 35:WRKY6-9 plants expressing the GUS reporter gene WRKY6-13 plants. driven by W - and W -containing promoter fragments, only weak Y Z We further tested if WRKY6 binding to PHO1 W-box motifs GUS staining can be detected (Figures 4B, 4C, and 4E). The was required for its function in regulation of PHO1 transcription. results demonstrate that binding to PHO1 W-box motifs was Different truncated PHO1 promoter fragments (indicated above required for WRKY6 regulation of PHO1 transcription. All these each panel of Figures 4B to 4D) driving the b-glucuronidase data support the notion that WRKY6 is the negative regulator of (GUS) reporter gene were transformed into 35S:WRKY6-9, PHO1 transcription. wrky6-1, and wild-type plants. In wild-type plants, the GUS reporter gene was expressed when driven by PHO1 promoter Repression of PHO1 Transcription by WRKY6 Is Removed fragment containing all four W-box motifs (W ,W ,W , and W ), Q X Y Z under Low Pi Stress two W-box motifs (W and W ), or no W-box motifs, respectively Y Z (Figures 4B to 4D). When all four W-box motifs were deleted from Consistent with previous reports (Stefanovic et al., 2007; Ribot the PHO1 promoter, the expression of the reporter gene in 35S: et al., 2008), we observed that PHO1 transcription was induced WRKY6-9 and wild-type roots was dramatically increased, indi- in response to low Pi stress. The PHO1 transcription level in 3558 The Plant Cell tion could be weakened. Protein gel blot analysis was performed using anti-WRKY6 serum in the total proteins extracted from the roots of seedlings grown on the low Pi medium. As shown in Figure 7A, the low Pi treatment induced a time-dependent decrease of WRKY6 protein content. Yeast two-hybrid assays (see Supplemental Table 1 online) showed that WRKY6 inter- acted with a RING-type finger E3 ligase (At1g74410), indicating that WRKY6 protein degradation may be mediated by the 26S proteosome. Addition of 10 mM MG132, a 26S proteosome inhibitor (Lee et al., 2009), blocked the low Pi–induced decrease of WRKY6 protein (Figures 7B and 7C), suggesting that a 26S proteasome–mediated WRKY6 proteolysis is involved in WRKY6-regulated PHO1 expression in response to low Pi stress. WRKY42 Interacts with the PHO1 Promoter and Negatively Regulates PHO1 Transcription Figure 3. ChIP Assays for At WRKY6 Binding to the W-Box of the PHO1 To identify other proteins that interact with WRKY6, we per- Promoter in Vivo. formed yeast two-hybrid experiments using WRKY6 as bait in (A) Diagram of the PHO1 promoter region showing the relative positions fusion with the Gal4 DNA binding domain. As listed in Supple- of four of six W-boxes (Q, 1718 to 1625; X, 1269 to 1181; Y, 966 mental Table 1 online, there are at least a dozen proteins that to 936; and Z, 775 to 618). W-boxes are marked by black rectan- interact with WRKY6. Among these WRKY6 interacting proteins, gles, and the untranslated region and exons of PHO1 are marked by gray WRKY42, as the closest homolog of WRKY6 (Eulgem et al., boxes. 2000), may have similar function to WRKY6. To test this hypoth- (B) ChIP-qPCR analysis of the PHO1 promoter sequence. ChIP assays were performed with chromatin prepared from wild-type Arabidopsis esis, we tested possible binding of WRKY42 with the PHO1 roots. The gray and black bars represent the ChIP signals with (WRKY6) promoter. The ChIP-qPCR experiments showed that, similar to and without (NoAB) addition of anti-WRKY6 serum, respectively. The WRKY6, WRKY42 can bind to both the Y and Z W-box motifs experiments were repeated three times, and three replicates were within the PHO1 promoter but not to the Q and X W-box motifs included for each sample in each experiment. The data are presented (Figure 8A). To further test possible function of WRKY42 on as means 6 SE (n =3). regulation of PHO1 expression, transient expression experi- ments in tobacco leaves were performed. The results showed wild-type roots was increased after the plants had been trans- that, similar to WRKY6, WRKY42 inhibited PHO1 promoter ferred to the low Pi medium for 3 d (Figure 5B; Ribot et al., 2008). activity (Figure 8B). The coinjection of Super:WRKY6 and Su- It was further proposed that the low Pi stress might trigger the per:WRKY42 showed much stronger repression on ProPHO1: plant responses through suppression of WRKY6 expression. GUS expression than did injection of either Super:WRKY6 or However, as shown in Figure 5A, after wild-type plants were Super:WRKY42 alone (Figure 8B). However, WRKY75 had no challenged with low Pi stress, WRKY6 expression level was effect on PHO1 expression and did not influence the inhibition of increased during the first 3 h and then decreased, but stayed PHO1 expression by WRKY6 (Figure 8B). above its zero time expression level for ;48 h. Another hypoth- Taking all these results together, we concluded that WRKY6 esis we proposed was that the Pi starvation inhibits WRKY6 functions in plant responses to low Pi stress by negatively functioning in suppression of PHO1 expression, such as through regulating PHO1 expression. Under normal conditions with suf- a possible blockage of WRKY6 binding to W-box motifs of the ficient Pi supply, WRKY6 (and probably also WRKY42) can bind PHO1 promoter or a low Pi–induced WRKY6 protein degrada- to the W-box motifs W and W within the PHO1 promoter and Y Z tion. ChIP-qPCR experiments were conducted to test whether represses the transcription of PHO1. Under Pi-deficient condi- WRKY6 protein still can bind to W and W boxes of the PHO1 Y Z tions, WRKY6 protein content is decreased via a 26S proteo- promoter under the low Pi condition. As shown in Figure 5C, the some–mediated proteolysis, and the interaction of WRKY6 and interaction between the WRKY6 protein and W or W box of the Y Z the PHO1 is limited. As a result, repression of PHO1 transcription PHO1 promoter was severely impaired under the low Pi condi- by WRKY6 is relieved, which might be important for plant tion. To confirm further the interaction of WRKY6 with the W and adaptation to a Pi-deficient environment. W boxes of the PHO1 promoter, ChIP-qPCR experiments were performed using the wild type, wrky6-1 mutant, and three WRKY6-overexpressing lines. As shown in Figure 6, the strong DISCUSSION interaction of WRKY6 with W or W boxes of PHO1 promoter Y Z was displayed again in all three WRKY6-overexpressing lines Plant-specific WRKY transcription factor family proteins have under the normal conditions (on MS medium), while this interac- been implicated in the regulation of genes involved in plant tion was reduced under the low Pi condition (on LP medium). responses to biotic as well as abiotic stresses, such as patho- It was further proposed that low Pi stress may induce degra- gen-induced stress (Dong et al., 2003; Eulgem and Somssich, dation of WRKY6 protein so that repression of PHO1 transcrip- 2007), drought, cold, and salinity stresses (Seki et al., 2002; Dong WRKY6 Regulates PHO1 Expression 3559 et al., 2003). WRKY factors act primarily by binding to conserved W-box elements in the promoters of specific targets to direct temporal and spatial expression of these genes (Ulker and Somssich, 2004). Among 74 members in the Arabidopsis WRKY family, only WRKY75 has been reported to be involved in modulation of Pi acquisition and root development (Devaiah et al., 2007a). This study demonstrated that WRKY6 (and prob- ably also WRKY42) plays an important role in modulation of plant responses to low Pi stress via regulation of PHO1 expression. WRKY6 Is a Negative Regulator for PHO1 Transcription Plant responses to Pi starvation involve the transcriptional reg- ulation of numerous genes to establish an adaptive mechanism (Franco-Zorilla et al., 2004). Several Arabidopsis transcription factors were identified functioning in the Pi starvation response, such as PHR1 (Rubio et al., 2001), ZAT6 (Devaiah et al., 2007b), BHLH32 (Chen et al., 2007), MYB62 (Devaiah et al., 2009), and a WRKY family protein WRKY75 (Devaiah et al., 2007a). WRKY75 can be induced by low Pi stress and is believed act as a positive regulator of Pi acquisition under Pi-deficient conditions (Devaiah et al., 2007a). In this study, we first observed that the WRKY6 overexpression lines displayed similar phenotypes as the pho1 mutant under low Pi stress, including growth inhibition and anthocyanin accumu- lation. We further demonstrated that WRKY6 protein can bind to two W-boxes of the PHO1 promoter and that PHO1 transcription was repressed by overexpression of WRKY6 under normal Pi supply conditions. This repression of PHO1 transcription by WRKY6 was relieved under low Pi conditions, indicating that the regulation of PHO1 transcription by WRKY6 is Pi dependent. In addition, protein blot analysis showed that the low Pi treatment– induced WRKY6 decrease was inhibited by a 26S proteosome inhibitor MG132. This suggests that the low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated WRKY6 proteolysis. Such a Pi-dependent mechanism may make WRKY6 a key regulator for plant responses to low Pi stress. Mechanism of PHO1 Regulation by WRKY6 and WRKY42 Under normal growth conditions, WRKY6 represses PHO1 ex- pression to balance Pi homeostasis through its binding to two W-boxes at the end of the coding region of the PHO1 promoter. When a low Pi stress signal is sensed by an unknown signaling mechanism and relayed to the E3 ubiquitin ligase, a 26S proteosome–mediated WRKY6 protein degradation is activated and WRKY6 binding to the PHO1 promoter W box motifs is weakened so that PHO1 transcription is induced to cope with the Figure 4. Suppression of PHO1 Expression by WRKY6. Pi-deficient environment. As a result, PHO1-facilitated Pi loading from root to xylem occurs and translocation of Pi from root to (A) RNA gel blot analysis of PHO1 expression in the roots of the WRKY6- overexpressing lines (Super:WRKY6-13, Super:WRKY6-18, and 35S: shoot could be promoted so that plants can adapt to a Pi- WRKY6-9), the wrky6-1 mutants, and wild-type plants. rRNA is shown as deficient environment. The plant may use WRKY6 (and probably a loading control. (B) to (D) GUS staining showing expression patterns of PHO1 in trans- genic plants carrying distinct PHO1 promoter constructs (indicated three independent transgenic lines for each background. All PHO1 above each panel; green boxes show W boxes, and yellow box repre- promoter–driven GUS transgenic lines are homozygous lines, and each sents the GUS gene) in 35S:WRKY6-9, wrky6-1 mutant, or wild-type line contains a single copy of insertion. backgrounds. The three roots in each group are representatives from (E) Relative GUS activities in different transgenic plants. 3560 The Plant Cell also WRKY42) as a key regulator that responds to varied Pi supply conditions and regulates Pi distribution in different organs via regulation of PHO1 as well as other unknown components. Our results also showed that, similar to WRKY6, WRKY42 can bind to both the Y and Z W-box motifs of the PHO1 promoter but not to the Q and X W-box motifs (Figure 8A). In addition, WRKY42 alone can also inhibit PHO1 expression (Figure 8B). Alternatively, considering that WRKY6 can interact with WRKY42 (see Sup- plemental Table 1 online), one may wonder if these two factors can form heterocomplexes to regulate PHO1 expression. Xu et al. (2006b) reported that three different kinds of WRKY proteins (WRKY18, WRKY40, and WRKY60) can interact with each other and form heterocomplexes, and the interactions between these WRKY factors influence their DNA binding activities. Robatzek and Somssich (2002) showed that WRKY6 can act as a negative regulator of its own and WRKY42 expression even though the mechanism and function remains unknown. To test possible synergic effects of WRKY6 and WRKY42 on PHO1 expression, we coinjected Super:WRKY6 and Super: WRKY42 in tobacco leaves to test their effects on ProPHO1: GUS expression. The results (Figure 8B) indicate that WRKY6 and WRKY42 together had stronger repression on PHO1 ex- pression. However, still we cannot conclude that they work together (by forming heterodimers) or work independently at this point. The ChIP-qPCR data showed that WRKY6 and WRKY42 had differential interactions with Y and Z W-boxes within the PHO1 promoter. WRKY6 displayed a stronger interaction with the Y box than with the Z-box (Figures 3B and 5C), while WRKY42 displayed a stronger interaction with the Z-box than with the Y-box (Figure 8A). These results indicate that WRKY6 and WRKY42 may regulate PHO1 expression in different ways. To clarify further if WRKY6 and WRKY42 work independently or together as a complex in regulation of PHO1 expression is an important issue for comprehensively understanding complex mechanisms of PHO1 regulation by WRKY factors. Regulation of PHO1 by Other Possible Regulatory Factors Although the Q and X W-boxes within the PHO1 promoter do not bind to WRKY6, we have noticed that the deletion of the sequences containing the Q- and X-boxes reduced the inhibitory effect of WRKY6 on PHO1 expression (Figures 4B, 4C, and 4E), particularly for wild-type plants. One of possible explanation for Figure 5. Repression of PHO1 Expression by WRKY6 Was Released in this phenomenon is that, within the deleted sequences, some Response to Low Pi Stress. other regulatory elements related to plant responses to low Pi stress may exist. The analysis of the deleted sequences using (A) qPCR analysis of WRKY6 expression induced by Pi starvation. TESS (http://www.cbil.upenn.edu/cgi-bin/tess/tess) shows that, (B) qPCR analysis of PHO1 expression induced by Pi starvation. (C) ChIP-qPCR assays to detect the association between WRKY6 and in addition to Q and X W-boxes, there are a number of regulatory W-boxes within the PHO1 promoter in wild-type plants under the normal elements for the following transcription factors: GATA factors, (MS) and LP conditions. The ChIP signals with (WRKY6) and without CCAAT-box transcription factor, multiprotein bridging factor (NoAB) addition of anti-WRKY6 serum are indicated. The data are 1 (MBF1), homeodomain-leucine zipper (HD-Zip) transcription presented as means 6 SE (n = 3). The experiments were repeated three factor, MYB transcription factor, Dof (DNA binding with one times, and three replicates were included for each sample in each finger) factor, heat shock transcriptional factor (HSF), etc. Al- experiment. though there is no report so far regarding PHO1 regulation by these TFs, at least two of them have been reported involving plant responses to low Pi stress. The HD-Zip factor has been reported to bind to the phosphate response domain of the soybean (Glycine max) VspB tripartite promoter (Tang et al., WRKY6 Regulates PHO1 Expression 3561 Figure 6. ChIP-qPCR Assays to Detect the Association of WRKY6 and the PHO1 Promoter in the Tested Plants as Indicated under Pi-Sufficient (MS) and Pi-Deficient (LP) Conditions. ChIP assays were performed with chromatin prepared from tested plants roots to analyze the binding of At WRKY6 protein to the W -box ([A]; Q site), W -box ([B]; X site), W -box ([C]; Y site), and W -box ([D]; Z site) of the PHO1 promoter in vivo. The ChIP signals with (WRKY6) and without (NoAB) X Y Z addition of anti-WRKY6 serum are indicated. The experiments were repeated three times, and three replicates were included for each sample in one experiment. The data are presented as means 6 SE (n = 3). 2001). Nilsson et al. (2007) reported that increased expression of et al., 1991; Hamburger et al., 2002). Considering these results the MYB-related transcription factor PHR1 resulted in enhance- together with the fact that WRKY75 did not have an effect on ment in phosphate uptake in Arabidopsis. It is plausible to further PHO1 promoter activity (Figure 8B; in addition, WRKY75 did not hypothesize that one or more of these regulatory elements (which interact with WRKY6 in the yeast two-hybrid assay), we may were deleted together with Q and X W-boxes in the experiments further hypothesize that WRKY75 and WRKY6, in response to shown in Figure 4C) may directly or indirectly be involved in low Pi stress, function in different regulatory pathways. Identify- PHO1 regulation. ing the gene(s) whose expression is specifically regulated by WRKY75 as well as other possible transcription factors will help us to clarify the complex mechanisms of plant responses to low WRKY6 and WRKY75 May Respond to Low Pi Stress via Pi stress. Different Pathways WRKY75 has been reported to play an important role in the Other Possible Roles of WRKY in Regulation of Pi Starvation phosphate starvation response, particularly by modulating Pi Responsive Genes uptake and root development (Devaiah et al., 2007a). The results presented here demonstrate that WRKY6 responds to low Pi To test if WRKY6 would play roles in regulation of other Pi stress by regulation of PHO1 transcription. Repression of starvation responsive genes, we performed comparative tran- WRKY75 expression (by RNA interference methods) resulted in scriptome analyses with various plant materials (35S:WRKY6-9, a decrease of Pi uptake (Devaiah et al., 2007a), while over- wrky6-1, and the wild type) using the Affymetrix GeneChip. As expression of WRKY6 repressed PHO1 expression and conse- shown in Supplemental Table 2 online, among 30 low Pi re- quently reduced Pi accumulation in shoots. It is known that sponse genes (Devaiah et al., 2007a; Lin et al., 2009), 11 of them PHO1 functions in Pi translocation from root to shoot (Poirier showed expression changes between either wild-type and 35S: 3562 The Plant Cell In addition, among the genes whose expression was upregu- lated or downregulated by more than two times and showed relevant changes (changes in opposite direction in 35S:WRKY6-9 plants compared with the wrky6-1 mutant), there are a total of 25 genes (listed in Supplemental Table 3 online) whose pro- moters contain W-box(es). Among these genes, there are 15 genes whose transcriptions were repressed, and transcription of 10 other genes was enhanced in 35S:WRKY6-9 plants. These data indicate that, in addition to its function in plant responses to low Pi stress, WRKY6 may be involved in a broad range of transcriptional regulations related to different processes, such as senescence, pathogen defense, and wounding responses (Robatzek and Somssich, 2001, 2002). Figure 7. WRKY6 Protein Blot Analysis. Seven-day-old wild-type seedlings were transferred to LP medium (A), LP medium with 10 mM MG132 (LP+MG132) (B), or LP medium with DMSO (LP+DMSO) (C). The roots of seedlings were harvested for protein extraction at the indicated time. Protein extracts were analyzed by immunoblots using rabbit anti-WRKY6 serum. Tubulin levels were detected in parallel as a loading control with antitubulin antibody. WRKY6-9 plants or the wild type and the wrky6-1 mutant. Among the members of the PHT1 family, Pht1;5 and Pht1;8 displayed transcriptional changes in 35S:WRKY6-9 plants and the wrky6-1 mutant compared with wild-type plants (see Supplemental Table 2 online). Both Pht1;5 and Pht1;8 contain W-boxes in their promoters (Devaiah et al., 2007), suggesting that WRKY6 may regulate their transcription. It is known that expression of Pht1;8 was significantly increased in the pho2 mutant (Aung et al., 2006; Bari et al., 2006), a mutant overaccumulating Pi in leaves, suggesting a possible role of Pht1;8 in WRKY6- and PHO1- Figure 8. Suppression of PHO1 Expression by WRKY42. related Pi mobilization. PS2 and PS3, two members of a phos- (A) ChIP-qPCR assays to detect the association between WRKY42 and phatase family, were significantly upregulated in 35S:WRKY6-9 W-boxes within the PHO1 promoter in wild-type plants under normal plants and downregulated in the wrky6-1 mutant (see Supple- conditions. The experiments were repeated three times, and three mental Table 2 online), suggesting that WRKY6 also might be replicates were included for each sample in each experiment. The data involved in plant early responses to low Pi stress (Devaiah et al., are presented as means 6 SE (n = 3). 2007). Several low Pi responsive transcription factors listed in (B) Transient overexpression of the ProPHO1:GUS fusion together with Supplemental Table 2 online, including PHR1, ZAT6, WRKY75, Super:WRKY6, Super:WRKY42,or Super:WRKY75 in Nicotiana ben- and BHLH32, did not show significant changes in their tran- thamiana leaves. ProPHO1:GUS fusion together with Super1300 vector scription either in 35S:WRKY6-9 plants or in the wrky6-1 mutant. was taken as the control. The data are presented as means 6 SE (n = 4). WRKY6 Regulates PHO1 Expression 3563 METHODS GATTTCAT-39 and the reverse primer 59-TTGGCGGCACCCTTACGTG- GATCA-39). EF1a was used as a quantitative control. For real-time PCR analysis, total RNA extraction was performed as Plant Materials and Growth Conditions described above, and the RNA was treated with DNase I RNase Free The WRKY6 overexpression lines 35S:WRKY6-3, 35S:WRKY6-5, 35S: (Takara) to eliminate genomic DNA contamination. The cDNA was syn- WRKY6-9, and the WRKY6 knockout mutant wrky6-1 were kindly pro- thesized from total RNA by SuperScript II RNase H reverse transcriptase vided by Imre E. Somssich (Max-Planck-Institut, Germany; Robatzek and (Invitrogen) using Radom Hexamer Primer (Promega). Quantitative real- Somssich, 2002). The Super:WRKY6-13 and Super:WRKY6-18 lines were time PCR was performed using the Power SYBR Green PCR Master Mix generated by cloning the coding sequence of WRKY6 into Super1300 (Applied Biosystems; P/N 4368577) on a 7500 Real Time PCR System vector (Li et al. 2001). The pho1 mutant was ordered from the ABRC machine (Applied Biosystems) following the manufacturer’s protocols. (http://www.Arabidopsis.org/abrc/). The PCR amplification was performed at 958C for 15 s and 608C for 1 min. For phenotype tests and seed harvest, Arabidopsis thaliana plants were Relative quantitative results were calculated by normalization to 18S grown in a potting soil mixture (rich soils:vermiculite = 2:1, v/v) and kept in rRNA. qPCR was conducted with WRKY6-specific primers (the forward 22 21 growth chambers at 228C with illumination at 120 mmol·m ·s for an primer 59-TAGTCACGACGGGATGATGA-39 and the reverse primer 18-h daily light period. The relative humidity was ;70% (65%). 59-ATTAGGAGGCGGAGGTGAGT-39)and PHO1-specific primers (the Low Pi stress treatment of plants was conducted by growing seedlings forward primer 59-TGGTTCTCCGGAACAAGAAC-39 and the reverse on Petri dishes containing Pi-sufficient (MS) or Pi-deficient (low Pi or LP) primer 59-TGACTTCAAGTGACGCCAAG-39). medium. The seeds were surface sterilized with the mixed solutions of NaClO (0.5%) and Triton X-100 (0.01%) for 10 min followed by washing Antibody Generation and ChIP-qPCR Assay with sterilized distilled water four times. The sterilized seeds were first The whole coding sequences were amplified by PCR using WRKY6- incubated on Petri dishes containing MS agar (0.8%) medium (containing specific primers (the forward primer 59-cccgggCCCGGGATGGACAGAG- 1.25 mM KH PO and 3% sucrose) at 48C for 2 d before germination. The 2 4 GAGGTCT-39 and the reverse primer 59-ctcgagCTCGAGCTATTGATTT- seeds were germinated at 228C under constant illumination at 40 22 21 TTGTTGTTTC-39)and WRKY42-specific primers (the forward primer mmol·m ·s and the 7-d-old seedlings were transferred to LP medium. 59-ggatccGGATCCATGTTTCGTTTTCCGGTAAG-39 and the reverse primer The LP medium was made by modification of MS medium such that the Pi 59-gagctcGTCGACTCTTATTGCCTATTGTCAAC-39). The lowercase let- (supplied with KH PO ) concentration in LP medium was 10 mM, and agar 2 4 ters represent the restriction sites. The whole coding sequences of At was replaced by agarose (Promega) to avoid the contamination of WRKY6 and WRKY42 were cloned into pGEX-4T-2 (Pharmacia). The phosphorous. reconstructed pGEX-4T-2 plasmid containing WRKY6 or WRKY42 was then transformed into Escherichia coli strain BL21 (DE3) to express Quantification of Total Pi proteins by induction with 0.1 mM isopropyl-b-D-thiogalactopyranoside Seven-day-old Arabidopsis seedlings germinated on MS medium were at 188C for 8 h. The resulting glutathione S-transferase (GST) fusion transferred to MS or LP medium for another 7 d, and then the roots and proteins were purified using Glutathione Sepharose 4B (Pharmacia). shoots were harvested for Pi content measurements. The samples were Protein concentrations were determined using the Bio-Rad protein assay oven dried at 808C for 48 h before determination of dry weight, and the kit. The polyclonal antibodies against WRKY6 or WRKY42, generated by samples were ashed in a muffle furnace at 3008C for 1 h and 5758Cfor an inoculation of rabbits, were purified using the Amino Link Plus Immobi- additional 5 h and then dissolved in 0.1 N HCl. The total Pi content in the lization Kit (Pierce). samples was quantified as described previously (Ames, 1966). The 7-d-old seedlings germinated on MS medium were transferred to MS or LP medium for another 7 d and then the roots were harvested for ChIP experiments. The chromatin samples for ChIP experiments were RNA Gel Blot, RT-PCR, and Real-Time PCR Analysis obtained following the methods by Saleh et al. (2008). The roots of plants For RNA gel blot analysis, 7-d-old Arabidopsis seedlings germinated on seedlings were first cross-linked by formaldehyde, and the purified cross- MS medium were transferred to MS or LP medium for another 7 d and linked nuclei were then sonicated to shear the chromatin into suitably then the seedlings or roots were harvested for extraction of total RNA sized fragments. The antibody that specifically recognizes the recombi- using the Trizol reagent (Invitrogen). Thirty micrograms of RNA were nant WRKY6-GST or WRKY42-GST was used to immunoprecipitate loaded per lane and transferred to a nylon membrane for hybridization. DNA/protein complexes from the chromatin preparation. The DNA in the Gene-specific probes were amplified by PCR using WRKY6-specific precipitated complexes was recovered and analyzed by qPCR methods. primers (the forward primer 59-CTTTGGCGATGTCTAGAATTGA-39 and Following the methods described by Haring et al. (2007), qPCR analysis the reverse primer 59-CCTCACCTACTGCTCTCGTAGG-39) and PHO1- was performed using the Power SYBR Green PCR Master Mix (Applied specific primers (the forward primer 59-TACTTGATTCTTTCTTACCC- Biosystems; P/N 4368577) in a 20-mL qPCR reaction on the 7500 Real TACTTCTGG-39 and the reverse primer 59-TCCAAGGAACGGTAACGG- Time PCR System machine (Applied Biosystems) following the manu- TACGGTCTTCACT-39) as the templates, respectively. The probes were facturer’s protocols. The chosen primer combinations (see Supplemental labeled with [a- P]dCTP using random primer labeling reagents (Phar- Table 4 online) can amplify fragments of 150 to 200 bp within the promoter macia) and hybridized to RNA gel blotted onto nylon membrane. The of PHO1. To ensure the reliability of ChIP data, the input sample and no- rRNA was taken as the control. antibody (NoAB) control sample were analyzed with each primer set. The For RT-PCR analysis, total RNA was extracted with Trizol reagent results were quantified with a calibration line made with DNA isolated (Invitrogen) and then treated with DNase I RNase Free (Takara) to from cross-linked and sonicated chromatin. eliminate genomic DNA contamination. The cDNA was synthesized from treated RNA by SuperScript II RNase H reverse transcriptase Vector Construction and Arabidopsis Transformation and (Invitrogen) using oligo(dT) primer (Promega). The PCR experiments GUS Assays were conducted with WRKY6-specific primers (the forward primer 59-ATGTTTCGTTTTCCGGTAAGTCTTGGAGGA-39 and the reverse PHO1 promoter variants, including the full-length promoter (2282 bp), the primer 59-TATTGCCTATTGTCAACGTTGCTCGTTGTAACATTA-39)and promoter containing two W-boxes (Y and Z, 1141 bp), and the promoter EF1a -specific primers (the forward primer 59-ATGCCCCAGGACATCGT- without W-box (454 bp), were amplified by PCR from Arabidopsis 3564 The Plant Cell genomic DNA and cloned into the transformation vector pCAMBIA1381 1.0 (MAS 5.0) software to generate detection calls and normalized using at the SalIand PstI restriction sites, respectively. All primer sequences Affymetrix GCOS software, and the TCT value was set to 100. When used for vector constructions are listed in Supplemental Table 5 online. All analyzing the transcriptionally changed genes, the signal ratio between tested Arabidopsis plants (Col-0, 35S:WRKY6-9, and the wrky6-1 mutant) two plant materials was calculated to represent the fold change of this were transformed by these three constructed vectors, respectively, using gene for its transcription in the corresponding process, and the fold the Agrobacterium tumefaciens–mediated gene-transfer procedure change P value for each gene was obtained at the same time using GCOS (Clough and Bent, 1998). All transgenic lines used in this study are T3 software. A gene was considered to be transcriptionally changed when it homozygous plants with single copy insertion. The GUS staining and GUS was upregulated or downregulated more than onefold. The genes with P activity measurements were performed as described previously (Xu et al., values < 0.05 and fold changes > 1 are included in Supplemental Table 3 2006a). online. Accession Numbers SDS-PAGE and Protein Gel Blot Analysis Sequence data from this article can be found in the Arabidopsis Genome For MG132 treatment, 10 mM stock solution was made by dissolving Initiative or GeneBank/EMBL database under the following accession MG132 (Calbiochem) in DMSO, and the final MG132 concentration was numbers: WRKY6 (At1g62300), WRKY42 (At4g04450), WRKY75 10 mM. For the control of MG132 treatment, 1/1000 DMSO was added to (At5g13080), PHO1 (At3g23430), and EF1a (At5g60390). Microarray the medium. The 7-d-old seedlings germinated on MS medium were data from this article have been deposited with the National Center for transferred to low Pi (LP), low Pi with 10 mM MG132 (Calbiochem) Biotechnology Information Gene Expression Omnibus data repository (LP+MG132), or low Pi with DMSO (LP+DMSO) medium, and then the (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE18273. roots were harvested at the indicated time for immunoblot analysis. Total proteins were extracted according to the method of Saleh et al. (2008), Supplemental Data and 50 mg proteins of each sample were separated on a 10% SDS-PAGE and analyzed by protein gel blot according to Towbin et al. (1979). Mouse The following materials are available in the online version of this article. anti-tubulin (Sigma-Aldrich) (with 1:4000 dilution) and rabbit anti-WRKY6 Supplemental Figure 1. Phenotype Test of WRKY6-Overexpressing serum (with 1:10,000 dilution) were used as the primary antibodies, and Lines. goat anti-mouse (or anti-rabbit) peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson) (with 1:8000 dilution) was used as the Supplemental Figure 2. Pi Content Measurements in Various Plant secondary antibody. The membranes were visualized using a Super- Materials. Signal West Femto Trial Kit (Themor) following the manufacturer’s in- Supplemental Figure 3. Phenotype Comparison of the wrky6-1 Mu- structions. tant, 35S:WRKY6-9, and Wild-Type Plants under a Short-Day (10-h- Light/14-h-Dark) Condition. Transient Expression Assays in Nicotiana benthamiana Supplemental Table 1. Yeast Two-Hybrid Results with At WRKY6 as the Bait. The constructs of ProPHO1:GUS with Super1300, Super1300:WRKY6,or Super1300:WRKY42 were transformed into A. Agrobacterium (GV3101). Supplemental Table 2. Expression of Pi Starvation Responsive Agrobacterium cells were harvested by centrifugation and suspended in Genes in the 35S:WRKY6-9 Plant and the wrky6-1 Mutant. the solutions containing 10 mM MES, pH 5.6, 10 mM MgCl , and 200 mM Supplemental Table 3. Genes with W-Boxes Whose Expression Was acetosyringone to an optical density (600 nm) of 0.7, incubated at room Up- or Downregulated More Than Twofold in the 35S:WRKY6-9 Line temperature for 4 h, and then used to infiltrate leaves of N. benthamiana (OE) and the wrky6-1 Mutant (KO) Compared with Wild-Type Plants. using a needle-free syringe. The GUS activity of the infiltrated leaves was quantitatively determined. Supplemental Table 4. Primers Used for ChIP-qPCR Experiments. Supplemental Table 5. Primers Used for the ProPHO1:GUS Con- Yeast Two-Hybrid Assay structs. The WRKY6 full-length cDNA sequence was amplified with WRKY6- specific primers (the forward primer 59-GCcatatgATGGACAGAG- ACKNOWLEDGMENTS GATGGTCTGG-39 and the reverse primer 59-GCgtcgacCTATT- This work was supported by a competitive research grant (30421002) for GATTTTTGTTGTTTCCTTCG-39) and cloned into pGBK-T7 vector at Creative Research Groups sponsored by the National Science Founda- NdeI and SalI sites. The lowercase letters in the primers indicate restric- tion of China and the Programme of Introducing Talents of Discipline to tion sites. Two-hybrid screening and assays were performed as de- Universities (no. B06003). We thank Imre E. Somssich (Max-Planck- scribed (Kohalmi et al., 1997). Strain AH109 (BD Biosciences) of Institut fu¨rZu¨ chtungsforschung, Abteilung Biochemie, Germany) for Saccharomyces cerevisiae was used in all yeast two-hybrid experiments. kindly providing At WRKY6 overexpression lines 35S:WRKY6-3, 35S: WRKY6-5, and 35S:WRKY6-9 and the At WRKY6 knockout mutant Microarray Analysis (wrky6-1). 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The Plant CellOxford University Press

Published: Nov 24, 2009

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