A Peroxidase Contributes to ROS Production during ... - Cell Press

Molecular Plant

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Volume 3

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Number 2

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Pages 420–427

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March 2010

RESEARCH ARTICLE

A Peroxidase Contributes to ROS Production during Arabidopsis Root Response to Potassium Deficiency Min Jung Kima, Silvano Cianib and Daniel P. Schachtmana,c,1 a Donald Danforth Plant Science Center, 975 North Warson Road, St Louis, MO 63132, USA b Present address: Department of Biology, Washington University in St Louis, St Louis, MO 63130, USA c Present address: Monsanto Company, St Louis, MO 63017, USA

ABSTRACT Reactive oxygen species (ROS) play an important role in root responses to potassium deprivation by regulating the expression of the high-affinity K1 transporter gene AtHAK5 and other genes. Activation-tagged lines of Arabidopsis plants containing the AtHAK5 promoter driving luciferase were screened for bioluminescence under potassiumsufficient conditions. A member of the type III peroxidase family, RCI3, was isolated and when it was overexpressed by the activation tag, this led to the enhanced expression of luciferase and the endogenous AtHAK5. RCI3 was found to be upregulated upon potassium deprivation. Plants overexpressing RCI3 (RCI3-ox) showed more ROS production and AtHAK5 expression whereas the ROS production and AtHAK5 expression were reduced in rci3-1 under K1-deprived conditions. These results suggested that RCI3 is involved in the production of ROS under potassium deprivation and that RCI3mediated ROS production affects the regulation of AtHAK5 expression. This peroxidase appears to be another component of the low-potassium signal transduction pathway in Arabidopsis roots. Key words:

Abiotic/environmental stress; nutrition; signal transduction; Arabidopsis; ROS production; peroxidase.

INTRODUCTION Reactive oxygen species (ROS) play important roles in many signal transduction pathways mediating responses to pathogen infection, abiotic stress, developmental regulation, and programmed cell death in different cell types (Apel and Hirt, 2004; Beers and McDowell, 2001; Laloi et al., 2004). ROS also accumulate in response to nitrogen, phosphorus, and potassium deprivation (Shin et al., 2005; Shin and Schachtman, 2004). The induced ROS under potassium deprivation plays a role in triggering gene expression (Shin and Schachtman, 2004) and ROS have also been suggested to act as an upstream regulator of calcium signaling (Lebaudy et al., 2007; Li et al., 2006). Plants generate ROS by activating various oxidases and peroxidases (Allan and Fluhr, 1997; Bolwell et al., 2002; Schopfer et al., 2001; Wasilewska et al., 2008). The NADPH oxidase, known as the respiratory burst oxidase, catalyzes the production of superoxide by the one-electron reduction of molecular oxygen using NADPH as an electron donor (Apel and Hirt, 2004). Peroxidases have been proposed as alternative producers of ROS (Apel and Hirt, 2004; Bindschedler et al., 2006). Peroxidases catalyze the oxidoreduction of various substrates using H2O2. The peroxidase rather than NADPH oxidase

has been proposed as the major ROS producer in the French bean (Phaseolus vulgaris) suspension-cultured cells treated with a cell wall elicitor of Colletotrichum lindemuthianum, which is the fungus that causes anthracnose disease (Bolwell et al., 1999, 1998). In Arabidopsis, the largest gene family of K+ transporters is the AtKT/KUP family of 13 genes (Very and Sentenac, 2003). Previously, we observed that AtHAK5 was the only gene in this family that was up-regulated upon K+ deprivation and rapidly down-regulated with resupply of K+ (Ahn et al., 2004). Based on the regulation of AtHAK5 gene expression, we set up a screening system that uses the luciferase reporter gene driven by the AtHAK5 promoter. This system was used to identify factors that are required for the potassium deprivationinduced response of the AtHAK5 transporter gene. Here, we

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To whom correspondence should be addressed at address a. E-mail [email protected], fax 1-314-587-1521, tel. 1-314-587-1421.

ª The Author 2010. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS. doi: 10.1093/mp/ssp121, Advance Access publication 5 February 2010 Received 2 November 2009; accepted 27 December 2009

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report the isolation and molecular characterization of a member of a family of peroxidases, named RCI3 (for Rare Cold Inducible gene 3). Expression analysis showed that RCI3 is induced upon K+ deprivation and overexpression of RCI3 increased AtHAK5 gene expression. Moreover, Arabidopsis overexpressing RCI3 had enhanced ROS production in roots. Previously, we observed that AtHAK5 gene expression is dependent on the ROS production (Jung et al., 2009; Shin and Schachtman, 2004). On the basis of these results, the function of RCI3 in root responses to low potassium is proposed.

RESULTS RCI3 Is a Positive Regulator of AtHAK5prom–LUC RCI3 was isolated by screening activation-tagged plants containing the AtHAK5prom–LUC reporter gene. The activationtagged lines were screened under K+-sufficient conditions when AtHAK5 reporter expression was normally repressed and 44 plants exhibiting strong luciferase activity were transferred to soil. One of the mutants identified from this screening, 8–1–24, showed high luminescence (Figure 1A) and enhanced endogenous AtHAK5 gene expression under K+sufficient conditions (data not shown).

Figure 1. Overexpression of the RCI3 Gene Activates the AtHAK5prom–LUC Reporter. (A) The 8–1–24 mutant plants emit strong luminescence under K+sufficient conditions. # 8–1–24, plants transformed with AtHAK5prom–LUC and tagged by pDKS2-7 activation vector. Negative control (NC), plants transformed with AtHAK5prom–LUC. Positive control (PC), plants transformed with 35Sprom–LUC (i.e. LUC under the control of 35S promoter). Left, bright-field image of all plants; right, luminescence image of the plants. The color scale on the right shows the luminescence intensity from dark blue (lowest) to white (highest). (B) The T-DNA inserted upstream of RCI3 (At1g05260) gene in the 8– 1–24 mutant. The GenomeWalker system was used to identify the TDNA insertion site with left border-specific primers. The distance from the T-DNA insertion site to each gene is indicated in kilobases. (C) RCI3 is overexpressed in the 8–1–24 mutant. Expression levels relative to the wild-type of two genes, At1g05260 and At1g05230, were obtained by quantitative real-time PCR in wildtype (WT), NC, and 8–1–24 mutant. Data represent the mean of three biological replicates.

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To identify the gene responsible for the activation of AtHAK5prom–LUC expression, we located the T-DNA insertion in the 8–1–24 mutant line. This was done using a genome walker approach with left border (LB)-specific primer sets. Sequencing and subsequent BLAST analysis of a portion of the cloned DNA indicated that the T-DNA inserted between the At1g05230 and At1g05260 genes. The closest open reading frame to this cloned fragment was At1g05260 and it was located ;4.0 kb from the 5’ end of the T-DNA LB (Figure 1B). To identify overexpressed genes in the vicinity of where the activation tag inserted, we used qRT–PCR to assay the expression of the genes at a distance of 12 kb on each side of where the activation tag inserted in the 8–1–24 mutant (Figure 1C). The open reading frame of At1g05230 was not differentially expressed in the wild-type AtHAK5promreporter only (NC) control or in the activation-tagged mutant line (8–1–24) lines. However, the RCI3 (At1g05260) gene was expressed nearly 100-fold higher in 8–1–24 compared to controls (Figure 1C).

Overexpression of RCI3 in Arabidopsis Enhances ROS Production in the Root To determine whether the RCI3 acts as a ROS-detoxifying or generating enzyme, ROS was measured in the roots of the wild-type, two RCI3-ox lines, and an rci3-1 knockout with 5(and 6-) carboxy-2#,7#-difluorodihydrofluorescein diacetate (DFFDA) membrane-permeable fluorescent dye. ROS was mainly detected in the root hair-differentiation zone (RHDZ) (Jung et al., 2009) and the ROS levels were 1.5–3 times in the RCI3-ox compared to wild-type, but the levels of ROS in rci3-1 were similar to wild-type (Figure 2B and 2C). Under K+-deficient conditions, ROS significantly increased in RHDZ of wild-type roots, as has been previously shown (Jung et al., 2009; Shin et al., 2005). The increased ROS levels in the RCI3-ox were similar to wild-type but significantly lower in rci3-1 compared to wild-type (Figure 2C) under potassium-deprived conditions. The induced ROS production in RCI3-ox roots was completely blocked by DPI, which is an NADPH oxidase inhibitor (Figure 2D). The ROS levels in the RCI3 overexpression lines and the mutant indicate that RCI3 contributes to ROS production when Arabidopsis roots are deprived of potassium.

RCI3 Was Localized to ER and then Secreted to the Cell Wall Sequence analysis indicated that RCI3 includes an endoplasmic reticulum signal peptide at the N-terminus (signal peptide sequence: MNCLIAIALSVSFFLVGIVGPIQA) and lacks the carboxyterminal propeptide displayed by the vacuole-targeted peroxidases (Johansson et al., 1992). This suggested that RCI3 may be trafficked from the ER to the cell wall according to the intracellular trafficking pathway of secretary protein (Bednarek and Raikhel, 1992; Llorente et al., 2002). Therefore, we localized RCI3 fused to yellow fluorescent protein at the C-terminus of RCI3 (RCI3–YFP) in Arabidopsis protoplasts (Figure 3).

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RCI3–YFP was localized to perinuclear areas and was visualized as small aggregates surrounding the nucleus and scattered in the cytoplasm after 24 h of incubation whereas the YFP control was distributed uniformly in the cytosol (Figure 3A). The reticulated pattern of fluorescence that represents the localization of RCI3 resembled the distribution of the endoplasmic reticulum (ER). To determine whether RCI3 is associated with the ER, sub-cellular co-localization was performed using an ER marker. For this assay, ER–CFP marker construct was obtained from the plant organelle marker stock center (Nelson et al., 2007; www.bio.utk.edu/cellbiol/markers/) and the plasmid was co-transfected with RCI3–YFP into Arabidopsis protoplasts. The RCI3–YFP overlapped with ER–CFP (Figure 3B). To determine whether RCI3 is secreted to the cell wall, we observed the cellular localization of RCI3–YFP by transient expression in onion epidermal cells after 48 h of incubation. As shown in Figure 3C, the RCI3–YFP was located in the cell wall, while the control YFP was uniformly distributed throughout the cell. The secretion of RCI3–YFP was confirmed in plasmolyzed onion cells (Figure 3D). Thus, we concluded that RCI3

Figure 3. RCI3–YFP Protein Is Localized to ER and then Secreted to the Cell Wall. Figure 2. ROS Production Is Induced in Plants Overexpressing RCI3 under Normal Conditions. (A) RT–PCR analysis of RCI3 expression in wild-type, four RCI3-ox, and rci3-1 plants. (B) ROS fluorescence images are shown in wild-type, RCI3-ox, and rci3-1 roots grown on K+-sufficient (+ K, 1.75 mM K+) or K+-deprived (–K,0 mMK+) medium.ROSwas visualized by staining3-day-oldroots with 20 lM DFFDA. Yellow and red colors indicate higher ROS production as indicated in pseudocolor scale. Inset shows enlargement of RHDZ. White lines show the boundary of roots. Bar = 100 lm. (C) Quantified data from images shown in (B). Roots were deprived K+ for 24 h. Data represent the mean 6 standard deviation. Asterisks indicate a significant difference; n = 10 plants). (D) Three-day-old RCI3-ox seedlings were incubated with NADPH oxidase inhibitor (DPI) for 1 h under K+-sufficient (+K, 1.75 mM K+) conditions.

(A) Sub-cellular localization of YFP control, RCI3–YFP, and ER–CFP in Arabidopsis protoplast. Signals were detected 24 h after incubation from bright-field (bright), YFP, or CFP. (B) Sub-cellular co-localization of RCI3–YFP with ER–CFP in Arabidopsis protoplast. Expression of the genes was examined at 24 h after transformation. Signals from bright-field (bright), YFP (YFP; green), CFP (CFP: red), and merge (co-localization appears yellow). (C) Fluorescence proteins were transiently expressed in onion epidermal cells: YFP (top) and RCI3–YFP (bottom). Signals were detected 48 h after incubation from bright-field (bright), YFP (YFP), and merged images. (D) The localization of RCI3–YFP in plasmolyzed onion cells: YFP (top) and RCI3–YFP (bottom). Signals were detected 48 h after incubation from bright-field (bright), YFP (YFP), and merged images. White lines show the boundary of plasma membrane of the cell.

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is a secreted protein and it is sorted to the cell wall through intracellular trafficking mechanism.

Expression of RCI3 Is Correlated with Low-Potassium Conditions Gene expression studies showed that RCI3 is up-regulated under abiotic stresses such as cold, dehydration, high salt, and ABA treatment (Llorente et al., 2002). To determine whether RCI3 expression is also up-regulated by nutrient deficiency, real-time PCR was performed on cDNA derived from RNA isolated from roots that had been deprived of potassium, nitrogen, or phosphorus. RCI3 transcript increased significantly in roots starved of K+ for 6 h (Figure 4A) but not by nitrogen or phosphorus deprivation. To gain insight into what functional processes RCI3 may influence, RCI3 was overexpressed in Arabidopsis under the control of a constitutive promoter and a homozygous T-DNA inactivation line rci3-1 (SALK_140204C) was isolated in which the T-DNA was integrated into the first exon region of RCI3. AtHAK5 expression was assayed in wild-type, two RCI3-ox and rci3-1 plants under complete nutrient and K+-deprived conditions (Figure 4B). Roots of plants overexpressing RCI3 showed two to three-fold greater levels of AtHAK5 gene expression compared to wild-type controls under complete nutrient conditions. AtHAK5 expression is normally induced by K+ deprivation in the wild-type (Ahn et al., 2004; Jung et al., 2009). The induction of AtHAK5 expression under K+ deprivation was similar in RCI3-ox compared to wild-type, whereas AtHAK5 expression was reduced around five-fold in the rci3-1 knockout allele (Figure 4B). Although the AtHAK5 transcript level was significantly reduced in rci3-1, K+ deprivation could still enhance AtHAK5 expression 2.5-fold in this mutant. These results indicate that RCI3 is sufficient to activate AtHAK5 expression but is not absolutely required.

Figure 4. RCI3 Expression Is Induced by K+ Deprivation and Affects AtHAK5 Expression. (A) Quantitative real-time PCR analysis of RCI3 under nutrient-deprived conditions. Arabidopsis Col-0 roots were grown under N (–N), P (–P), or K+ deficiency (–K) for 6 or 30 h. Asterisks indicate a significant difference as compared to other means in each subfigure (* p , 0.05; Student’s t-test). (B) AtHAK5 expression in wild-type, RCI3-ox, and rci3-1 knockout plants under K+-sufficient or K+-deprived conditions. RNA was extracted from whole plants. Data represent the mean of three biological replicates. Different letters indicate a significant difference between means at p , 0.05 (TUKEY HSD).

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RCI3 Affects Primary Root Growth but Not Tolerance to Low K+ More AtHAK5 expression was detected in RCI3 overexpressing plants than wild-type controls under complete nutrient conditions (Figure 4B). Previously, we found that AtHAK5 functions in root growth regulation (Qi et al., 2008). To determine whether RCI3 is also involved in the regulation of root growth, primary root length was measured in wild-type, RCI3-ox, and rci3-1 plants under K+-sufficient and K+-deprivation conditions. As shown in Figure 5, the primary roots of both RCI3-ox and rci3-1 are significantly smaller compared to wild-type control roots under K+-sufficient conditions (Figure 5B). To compare the root growth under low-K+ conditions, the percentage of primary root length under K+-sufficient conditions versus deprived conditions was calculated (Figure 5D). The RCI3-ox and rci3-1 mutants showed a similar growth pattern to wild-type control plants. We also measured the number of lateral roots and calculated lateral root density (number of laterals per length of primary root). The lateral root density of RCI3-ox and rci3-1 was the same as wild-type plants (Supplemental Figure 1A). RCI3-ox and rci3-1 shoot fresh weight and total chlorophyll content were also measured (Supplemental Figure 1B) and not found to be different from the wild-type plants. These results indicate that RCI3 plays a role in root growth regulation under normal conditions but does not affect tolerance to low K+.

Figure 5. Altered RCI3 Expression Alters Primary Root Growth in K+Sufficient but Not K+-Deprived Conditions. (A, B) Plants were grown under complete nutrient conditions for 4 d and then transferred to either full nutrient (A, +K) or a medium with no potassium (B, –K) for 7 d. (C) Primary root length of plants shown in (A) and (B) was measured. Data represent the mean 6 standard deviation. Different letters indicate a significant difference between means at p , 0.05 (TUKEY HSD) (n = 70 plants). (D) The primary root-length as A percentage of full nutrient medium to medium without potassium. Data represent the mean 6 standard deviation. Different letters indicate a significant difference between means at p , 0.05 (TUKEY HSD) (n = 70 plants).

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DISCUSSION Screening of activation-tagged plants with AtHAK5prom–LUC reportergeneconstructledtotheisolationofRCI3. RCI3isknown as a cold-inducible gene and suggested to be involved in tolerance to abiotic stresses such as dehydration and salt stresses (Llorente et al., 2002). RCI3 was induced under potassiumdeprivationconditions(Figure 4A) and RCI3 proteinwas secreted to the cell wall as previously predicted (Llorente et al., 2002) (Figure 3).RCI3-overexpressingplants showed more ROSproduction and more AtHAK5 gene expression compared to wild-type (Figures 2 and 4B). These findings suggest that RCI3 is a positive regulator in the ROS production and activation of AtHAK5 gene expression under potassium-deprivation conditions.

RCI3 Is Involved in ROS Production RCI3 is a member of class III peroxidases (Welinder et al., 2002) and has peroxidase activity only in the root cell (Llorente et al., 2002). It is known that class III peroxidases are targeted via the endoplasmic reticulum (ER) to the outside of the plant cell or to the vacuole (Welinder et al., 2002) and the cell wall-localized peroxidases play a role in ROS production (Bindschedler et al., 2006; Choi et al., 2007; Pnueli et al., 2003). Transgenic Arabidopsis plants expressing an anti-sense French bean peroxidase type 1 (FBP1) cDNA exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens (Bindschedler et al., 2006). The overexpression of CaPO2, a novel extracellular peroxidase gene from pepper (Capsicum annuum), in Arabidopsis showed increased H2O2 concentrations in response to Pst DC3000 infection (Choi et al., 2007). In RCI3-ox, more ROS were produced under full nutrient conditions than in the wild-type and the ROS level induced by potassium deprivation (Shin et al., 2005) was significantly decreased in rci3-1 compared to wild-type (Figure 2B and 2C). These data show that RCI3 is involved in ROS production.

RCI3 Is a Component of the Potassium-Deprivation Response Pathway A previous study showed that potassium deprivation stimulates ethylene production that then induces an increase in ROS production (Shin et al., 2005; Shin and Schachtman, 2004). Recently published studies also implicate calcium in the low-potassium signaling pathway (Li et al., 2006; Xu et al., 2006). Ethylene acts as an upstream factor involved in the initiation of ROS production. RCI3 was up-regulated under low-potassium conditions (Figure 4A) and RCI3-ox plants showed more AtHAK5 expression under full-nutrient conditions and the induction of AtHAK5 expression by low K+ was significantly reduced in rci3-1 plants (Figure 4B). Previously, we demonstrated that ROS and ethylene production increase in plant roots in response to potassium deprivation and that low K+-inducible AtHAK5 expression is dependent on ROS production (Jung et al., 2009; Shin and Schachtman, 2004).

These results suggest that RCI3 is a positive regulator of AtHAK5 gene expression during response to low potassium. Previously, we showed that H2O2 production under potassium deprivation is NADPH oxidase-dependent (Shin et al., 2005) and that diphenylene iodonium (DPI), an NADPH oxidase inhibitor, completely inhibited low-K+-induced AtHAK5prom– LUC expression (Jung et al., 2009). In this study, we showed that the induced ROS production in RCI3-ox roots was completely blocked by DPI treatment (Figure 2D). DPI has been used frequently as a specific inhibitor of NADPH oxidase activity and it has been reported that the peroxidase-mediated oxidative burst is not sensitive to DPI (Bolwell et al., 1998). However, another detailed study showed that DPI inhibits the H2O2 production by horseradish peroxidase (Frahry and Schopfer, 1998) and concluded that DPI may not effectively be used to distinguish between NADPH oxidase and peroxidase activity. Peroxidases may be an initial source of ROS that activates NADPH oxidases, which, in turn, generate a plasma membraneassociated oxidative burst (Bindschedler et al., 2006; Torres and Dangl, 2005). However, based on the nature of the effects of DPI (Frahry and Schopfer, 1998), we cannot conclude whether the peroxidase described in our work acts upstream or downstream of an NADPH oxidase. Ethylene signaling is known to be a component of plant responses to low potassium that stimulate the production of ROS (Jung et al., 2009; Shin and Schachtman, 2004). To identify whether ethylene is an up-stream factor of RCI3, we assayed the expression of RCI3 after 100 lM ethephon treatment in the seedling but the expression was not changed (data not shown). Previous studies showed that RCI3 expression was induced by ABA treatment in etiolated seedlings but not in roots (Llorente et al., 2002). The up-regulation of RCI3 by ABA further supports the previous finding that ABA is involved in the plant response to low potassium (Kim et al., 2009) and we speculate the ethylene and ABA may provide two parallel pathways for response to low potassium. The influence of RCI3 expression in tolerance to low potassium was estimated by comparing plant root and shoot growth. Transgenic plants overexpressing RCI3 showed similar growth to wildtype plants (Figure 5 and Supplemental Figure 1B). The lack of a low-potassium phenotype may be due to the complexity of how growth is regulated. It also indicates that overexpression of RCI3 is not sufficient to overcome the stress conditions imposed by potassium deprivation, even though RCI3 is induced by low K+ andissufficientforROSproductionandAtHAK5expression.Taken together, our data suggest that RCI3-mediated ROS production is a new component involved in the regulation of AtHAK5 gene expression and overall response to low-potassium conditions. We suggest that RCI3 could function downstream of ABA production in the low-K+ signaling pathway (Kim et al., 2009; Peuke et al., 2002), which activates RCI3 expression and participates in activating ROS production in the root hair differentiation zone. The activity of RCI3 participates in the induction of the high-affinity K+ uptake transporter AtHAK5, which ultimately facilitates the acquisition of potassium under conditions of deprivation.

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METHODS Generation of Activation-Tagged Mutant Plants with AtHAK5prom–LUC Reporter Gene Construct Activation-tagged mutant plants with the AtHAK5 (At4g13420) promoter fused to a LUC transgene construct were generated as previously described (Jung et al., 2009). AtHAK5prom–LUC homozygote plants were transformed with the activation tagging construct, pDKS 2–7 vector (Turk et al., 2005). M1 seeds were selected on plates containing nutrient solution previously described (Shin et al., 2005) with the addition of 2% sucrose, 0.6% SeaKem agarose (Cambrex), and 50 mg L 1 of Kanamycin. The 6000 kanamycin resistant seedlings were transferred onto K+-sufficient medium (contains 1.75 mM KCl) for 6 d under light and then sprayed with luciferin and detected fluorescence imaging with the 7383 CCD camera (Roper Scientific). Finally, images were converted into pseudocolor images using the NIH ImageJ software. The luciferase activities of at least two independent experiments were performed, and similar results were obtained.

Identification of T-DNA Insertion Sites in ActivationTagged Mutant Lines To identify the T-DNA insertion site, a genome walker system was used. The Arabidopsis libraries were prepared by digesting the DNA with four blunt-ended enzymes and then ligated to specific adapters according to the method in the Universal Genome Walker Kit user manual (CLONTECH). Two gene-specific primers (GSP1 and GSP2) were designed using the pDKS 2–7 vector sequence: LB-GSP1 5#-CATGTAGATTTCCCGGACATGAAGCCATTTAC-3’ and LB-GPS2: 5#-ATCCTGCCGCCGCTGCCGCTTTGCAC-3#. The fragments resulting from the PCR reactions performed on the Genome Walker libraries were sequenced; 5’ sequences upstream of the LB region were analyzed.

RCI3 DNA Cloning and Plant Transformation The RCI3 (At1g05260) open reading frame was amplified using Pfu-polymerase (Stratagene) and the following primers: 5#- ACACCATGAATTGCTTGATAGCTATAGCTC-3’ and 5#- ACTCGAGTTCCACCCCTTAACTATTTGCAA-3#. Transgenic plants carrying constitutively overexpressing RCI3 (RCI3-ox) were generated. For constitutive expression, we used pEarleygate100 vector with 35S CaMV promoter (Earley et al., 2006). Plants were transformed using Agrobacterium tumefaciens by the floral dip method (Clough and Bent, 1998). Two overexpressing lines with high RCI3 expression were chosen for further analysis.

Plant Material and Growth Conditions The Arabidopsis thaliana Columbia ecotype (Col-0) was used for this study. The homozygote rci3-1 T-DNA insertion line (SALK_140204C) used in this study was obtained from the SALK collection and the absence of RCI3 transcript was confirmed by

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reverse transcription PCR. Prior to growth assays, the plants were grown together under the same conditions to generate wild-type, overexpression, and knockout seed for further experiments. For K+-deficiency growth assays, 0 lM KCl was used instead of 1.75 mM KCl and the plants were grown on plates containing nutrient solutions as previously described (Shin et al., 2005) with the addition of 2% sucrose and 0.6% SeaKem agarose (Cambrex). After stratification of the seeds at 4C for 2–3 d, the plates were transferred to the growth chamber at 22C with a 16-h day length at 200 lmol m 2 s 1. Four-day-old seedlings were transferred to nutrient deficient medium and then the lengths of primary roots were measured 7 d after the transfer in three independent experiments with 70 plants per genotype and experiment.

ROS Detection and Measurement To detect ROS, 2-day-old seedlings were floated on K+-sufficient and K+-deficient liquid nutrient solutions and incubated with 20 lM DFFDA (Invitrogen) for 20 min. The roots were washed with liquid nutrient solution before visualizing the fluorescence with microscopy (Jung et al., 2009). All fluorescence images were captured using a Nikon SMZ1500 microscope and a Q-Imaging Retiga cooled 12-bit camera. ROS fluorescence in the root hair elongation zone (0.5 mm) was quantified and converted into pseudocolor images by the NIH ImageJ software program. Background noise was subtracted from the fluorescence intensity value for quantification. The diphenylene iodonium (DPI, Sigma) stock solution was prepared in 100% DMSO and roots were incubated in 10 lM DPI for 40 min before DFFDA treatment and during the 20-min DFFDA incubation for a total of 1 h. Three experiments on independently grown plant material were performed to confirm the results.

Quantitative Real-Time PCR Analysis The analysis of the gene expression with real-time PCR was performed as previously described (Kim et al., 2009). To analyze the expression of RCI3, the gene-specific primers (5#-GGAAATGTAAAAGATTTGGGAGCTTG-3’ and 5#-TATTGACGAAGTCTGTAACAAAATCC-3’) were used. b-tubulin gene (At5g62690) was amplified with the primers 5#-GCCAATCCGGTGCTGGTAACA-3’ and 5#-CATACCAGATCCAGTTCCTCCTCCC-3’) and was used to normalize the reactions. Real-time PCR was performed according to the instructions provided for the Bio-Rad iCycler iQ system (Bio-Rad laboratories) with platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). The fold change of transcripts was calculated based on an efficiency-calibrated model (Yuan et al., 2006) andcomparedwiththetranscript level under normalconditions. Statistical differences between samples were evaluated by Student’s t-test using delta Ct values (Yuan et al., 2006). In each experiment, the mean of three biological replicates was used to generate means and statistical significance. At least two experiments on independently grown plant material were performed to confirm the results.

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Visualization of RCI3–YFP Localization The RCI3–YFP constructs were introduced into Arabidopsis protoplasts, prepared from whole seedlings by the polyethylene glycol-mediated transformation procedure (Jin et al., 2001). For transient expression of RCI3–YFP, biolistic bombardment on onion epidermal cells was performed as previously described (Marc et al., 1998) using 5 lg of plasmid DNA precipitated onto gold particles. Plasmolysis of the onion epidermal cell was induced by the addition of 0.8 M mannitol (Friedrichsen et al., 2000) and imaged using a Nikon Eclipse E800 microscope. Two experiments on independently grown plant material were performed to confirm the results. The ER–CFP marker construct was obtained from the plant organelle marker stock center (stock number: CD3-953) that was created by combining the signal peptide of AtWAK2 at the N-terminus of the FP and the ER retention signal His-ASPGLU-Leu at its C-terminus (Nelson et al., 2007).

SUPPLEMENTARY DATA

Bolwell, G.P., Bindschedler, L.V., Blee, K.A., Butt, V.S., Davies, D.R., Gardner, S.L., Gerrish, C., and Minibayeva, F. (2002). The apoplastic oxidative burst in response to biotic stress in plants: a threecomponent system. J. Exp. Bot. 53, 1367–1376. Bolwell, G.P., Blee, K.A., Butt, V.S., Davies, D.R., Gardner, S.L., Gerrish, C., Minibayeva, F., Rowntree, E.G., and Wojtaszek, P. (1999). Recent advances in understanding the origin of the apoplastic oxidative burst in plant cells. Free Radic. Res. 31 Suppl, S137–S145. Bolwell, G.P., Davies, D.R., Gerrish, C., Auh, C.K., and Murphy, T.M. (1998). Comparative biochemistry of the oxidative burst produced by rose and French bean cells reveals two distinct mechanisms. Plant Physiol. 116, 1379–1385. Choi, H.W., Kim, Y.J., Lee, S.C., Hong, J.K., and Hwang, B.K. (2007). Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens. Plant Physiol. 145, 890–904. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743.

Supplementary Data are available at Molecular Plant Online.

Earley, K.W., Haag, J.R., Pontes, O., Opper, K., Juehne, T., Song, K., and Pikaard, C.S. (2006). Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 45, 616–629.

FUNDING

Frahry, G., and Schopfer, P. (1998). Inhibition of O2-reducing activity of horseradish peroxidase by diphenyleneiodonium. Phytochemistry. 48, 223–227.

We thank the Lubin Foundation for partial funding and Monsanto Company for the additional funding and for providing D.P.S. with the time to finalize this work.

ACKNOWLEDGMENTS We thank Christine Ehret for assistance in manuscript editing, Ji-Yul Jung for assistance in ROS detection, and Howard Berg for assistance in the RCI3–YFP localization test and Ryoung Shin for generating the promoter LUC lines. No conflict of interest declared.

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