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This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has beenedited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version onlinereduces the time to publication by several weeks.
The Arabidopsis Chaperone J3 Regulates the Plasma Membrane H+-ATPase through Interaction with thePKS5 Kinase C W Yongqing Yang,a,b,c,1 Yunxia Qin,d,1 Changgen Xie,a,b,1 Feiyi Zhao,e,1 Jinfeng Zhao,b Dafa Liu,d Shouyi Chen,eAnja T. Fuglsang,f Michael G. Palmgren,f Karen S. Schumaker,g Xing Wang Deng,a and Yan Guob,c,2 a College of Life Sciences, Peking University, Beijing 100871, Chinab National Institute of Biological Sciences, Beijing 102206, Chinac State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University,Beijing 100094, Chinad Key Lab of Ministry of Agriculture for Biology of Rubber Tree, Rubber Research Institute, Chinese Academy of TropicalAgricultural Sciences, Danzhou, Hainan 571737, Chinae Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101 Chinaf Department of Plant Biology, University of Copenhagen, DK-1871 Frederiksberg C, Denmarkg Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721 The plasma membrane H+-ATPase (PM H+-ATPase) plays an important role in the regulation of ion and metabolite transportand is involved in physiological processes that include cell growth, intracellular pH, and stomatal regulation. PM H+-ATPaseactivity is controlled by many factors, including hormones, calcium, light, and environmental stresses like increased soilsalinity. We have previously shown that the Arabidopsis thaliana Salt Overly Sensitive2-Like Protein Kinase5 (PKS5)negatively regulates the PM H+-ATPase. Here, we report that a chaperone, J3 (DnaJ homolog 3; heat shock protein 40-like),activates PM H+-ATPase activity by physically interacting with and repressing PKS5 kinase activity. Plants lacking J3 arehypersensitive to salt at high external pH and exhibit decreased PM H+-ATPase activity. J3 functions upstream of PKS5 asdouble mutants generated using j3-1 and several pks5 mutant alleles with altered kinase activity have levels of PM H+-ATPase activity and responses to salt at alkaline pH similar to their corresponding pks5 mutant. Taken together, our resultsdemonstrate that regulation of PM H+-ATPase activity by J3 takes place via inactivation of the PKS5 kinase.
(Palmgren, 2001; Rober-Kleber et al., 2003; Fuglsang et al.,2007; Gevaudant et al., 2007; Merlot et al., 2007). A number of In both plants and fungi, transport across the plasma membrane factors, including hormones (auxin and abscisic acid [ABA]), (PM) is energized by an electrochemical gradient of protons (H+).
calcium, blue light, and fungal elicitors, have been shown to elicit These gradients are established by the electrogenic PM H+ pumps changes in cellular pH through regulation of the PM H+-ATPase (ATPases), which convert chemical energy derived from hydrolysis (Kinoshita et al., 1995; Xing et al., 1997; Kim et al., 2001; Brault of ATP into pH and electrical gradients across the plasma mem- et al., 2004; Zhang et al., 2004). For example, auxin activates the brane (Palmgren, 2001). The combined electrochemical gradient H+-ATPase, resulting in apoplastic acidification, a process lead- constitutes a driving force for the transport of solutes and metab- ing to cell wall loosening and cell and organ elongation (Rober- olites across the plasma membrane (Morsomme and Boutry, 2000).
Kleber et al., 2003). Exogenous application of ABA on leaves has In Arabidopsis thaliana, PM H+-ATPases are encoded by a 12- an inhibitory effect on PM H+-ATPase activity (Roelfsema et al, member gene family (AHA1 to AHA12; Palmgren, 2001). These 1998; Zhang et al., 2004), while mutations in the PM H+-ATPase H+-ATPases play regulatory roles in signal transduction, during AHA1 (ost2) result in a constitutively active protein and plants cell expansion, turgor regulation, in the regulation of intracellular with reduced sensitivity to ABA during stomatal movement pH, and in the response of the plant to increases in soil salinity (Merlot et al., 2007). Evidence also exists linking PM H+-ATPaseactivity and increased salt tolerance as overexpression of an acti- 1 These authors contributed equally to this work.
vated PM H+-ATPase increased plant salt tolerance (Gevaudant 2 Address correspondence to guoyan@cau.edu.cn.
et al., 2007). This regulation appears to be due to posttransla- The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy described tional modification of the protein based on the fact that PM in the Instructions for Authors (www.plantcell.org) is: Yan Guo H+-ATPase protein levels do not change when plants are grown in salt (Ayala et al., 1996; Wu and Seliskar, 1998; Morsomme and C Some figures in this article are displayed in color online but in black Boutry, 2000).
and white in the print edition.
One well-characterized mechanism underlying regulation of Online version contains Web-only data.
PM H+-ATPase activity involves an autoinhibitory domain in The Plant Cell Preview, www.aspb.org ã 2010 American Society of Plant Biologists


Figure 1. PKS5 Interacts with J3.
(A) Schematic diagram of the PKS5, PKS5N, PKS5C, J3, J3C-219, J3C1, and J3C2 proteins used in the yeast two-hybrid analysis. KDAL, kinaseactivation loop; FISL, SCaBP interaction domain; PPI, phosphatase interaction domain; J-domain, DnaJ domain; G/F, domain rich in Gly and Pheresidues; C-rich domain, a distal zinc finger (CxxCxGxG)4 domain.
(B) to (D) Yeast two-hybrid analysis of interactions between the full-length PKS5 protein (PKS5) or the N- (PKS5N) or C- (PKS5C) terminal portions of theprotein with J3, or J3C-219, J3C1, or J3C2. Interactions between the full-length PKS5 protein or the protein with the FISL domain deleted (PKS5DF) and J3 Activates Plasma Membrane H+-ATPase Activity C-terminal region of the protein (Palmgren et al., 1991). Phos- evolution and is important for protein translation, folding, unfold- phorylation of this C-terminal autoinhibitory domain at the pen- ing, translocation, and degradation in a broad array of cellular ultimate residue (Thr-947) leads to its interaction with a 14-3-3 processes (Boston et al., 1996; Waters et al., 1996; Wang et al., regulatory protein and activation of the H+-ATPase (Svennelid 2004). Expression of Hsps in planta is induced by high temper- et al., 1999; Camoni et al., 2000; Gevaudant et al., 2007). The ature and also by a wide range of other environmental stresses, activated protein complex is likely to consist of six phosphory- including increased soil salinity and osmotic, water, cold, and lated PM H+-ATPase molecules assembled in a hexameric oxidative stresses (Boston et al., 1996; Waters et al., 1996; Wang structure together with six 14-3-3 molecules (Kanczewska et al., 2004). In addition to their function as chaperon proteins, et al., 2005; Ottmann et al., 2007). We recently demonstrated DnaJs are also involved in other biological processes, including that the PKS5 protein kinase negatively regulates the activity of regulation of transcriptional activation by directly binding tran- the PM H+-ATPase by directly phosphorylating the AHA2 isoform scription factors (Ham et al., 2006), formation of endosomes of the enzyme in its C-terminal regulatory domain at Ser-931 and (Tamura et al., 2007), and in carotenoid accumulation (Lu et al., that this phosphorylation inhibits the interaction between AHA2 2006). There are 89 putative J-domain proteins predicted in and the 14-3-3 protein (Fuglsang et al., 2007). A role for PKS5 in Arabidopsis (Miernyk, 2001). These J-domain proteins are both the regulation of the PM H+-ATPase was further supported by the soluble and found in membrane compartments of all cellular recent demonstration that Ser-938 (identical to Ser-931 in AHA2 organelles (Miernyk, 2001). J3 (Arabidopsis DnaJ homologous in Arabidopsis) is phosphorylated in vivo in PMA2, a PM-H+- protein3) contains all typical functional domains found in J-domain ATPase isoform in tobacco (Nicotiana tabacum; Duby et al., family members (Zhou and Miernyk, 1999). J3 is expressed in roots, stems, leaves, flower buds, flowers, and siliques, and its Environmental stresses in plants often cause protein denatur- expression can be induced by heat and water stress (Zhou and ation; therefore, maintaining proteins in their functional confor- Miernyk, 1999; Li et al., 2005).
mations and preventing protein aggregation are particularly In this study, we identify a DnaJ-like protein, Arabidopsis J3, as important for cell survival under stress conditions. Molecular a positive regulator of the PM H+-ATPase. We show that J3 chaperones (heat shock proteins [Hsps]) are key components interacts with and represses activity of the PKS5 kinase. To- contributing to cellular homeostasis under adverse growth con- gether with results from our genetic studies, we demonstrate that ditions (Wang et al., 2004). DnaJ/Hsp40 was originally charac- J3 regulates PM H+-ATPase activity through interaction with the terized in Escherichia coli as a 41-kD heat shock protein that PKS5 kinase.
interacts directly with DnaK and GrpE constituting a molecularchaperone machine (Georgopoulos et al., 1980; Liberek et al.,1991; Scidmore et al., 1993; Bukau and Horwich, 1998; Goffin and Georgopoulos, 1998; Miernyk, 1999). Additionally, DnaJ canact independently as a chaperone (Laufen et al., 1999). Most PKS5 Interacts with J3 DnaJ proteins contain a J-domain, a proximal G/F-domain, and adistal zinc finger (CxxCxGxG)4 domain, followed by less con- To understand how PKS5 regulates the PM H+-ATPase, we served C-terminal sequences (Caplan et al., 1993; Silver and identified PKS5-interacting proteins using yeast two-hybrid Way, 1993). The J domain, a 70–amino acid sequence, contains assays. To do this, we cloned the PKS5 cDNA into the pAS2 four helices and a highly conserved tripeptide made up of His, vector and transformed the resulting plasmid into yeast strain Pro, and Asp (the HPD motif) in the loop region between helices II Y190. PKS5 was then used as bait to screen an Arabidopsis and III (Qian et al., 1996). The J domain binds to Hsp70s, and this cDNA library (obtained from The Arabidopsis Information Re- binding stabilizes Hsp70 interaction with substrate proteins (Qiu source [TAIR]). Two positive clones were sequenced and found et al., 2006). The G/F-domain, which is rich in Gly and Phe to include 219 amino acids (J3C-219) that are identical to the C residues and comprises a flexible linker region, helps to confer terminus of At3g44110, which encodes a putative cochaperone interaction specificity among DnaK, DnaJ, and target polypep- DnaJ-like heat shock protein (J3) (Zhou and Miernyk, 1999). To tides (Wall et al., 1995; Yan and Yan, 1999). The distal zinc finger narrow down the interaction domain in J3, J3C-219 was divided domain is believed to participate in protein-protein interactions into two parts, J3C1 (amino acids 202 to 317) and J3C2 (amino among DnaJ, DnaK, and target polypeptides (Banecki et al., acids 316 to 420); the structures of the peptides are shown in 1996; Szabo et al., 1996). DnaJ has been conserved throughout Figure 1A. These fragments and the full-length J3 were cloned Figure 1. (continued).
SCaBP1 were used as positive and negative controls, respectively. Yeast lines harboring the indicated plasmids were grown on synthetic complete (SC)medium without Leu and Trp (SC-LW, left panel) and on SC medium without Leu, Trp, and His (SC-LWH) and with 20 mM 3-amino-1,2,4-triazole (3-AT;right panel). Yeast cells were incubated until OD660 = 1 and then diluted 10- or 100-fold and used for assays.
(E) Coimmunoprecipitation of PKS5 and J3 proteins in vivo. The 35SP:63Myc-J3 plasmid was cotransformed into wild-type (Col-0) protoplasts with35SP:33FLAG-PKS5 (lanes 1 and 3) or 35SP:33FLAG-TTG1 (lanes 2 and 4). Total protein extracts were analyzed with immunoblots using anti-Myc andanti-FLAG antibodies to detect the presence of PKS5, J3, or TTG1 (input). Immunoprecipitation was performed using anti-Myc agarose conjugate, andthe products were analyzed with immunoblots using anti-FLAG antibody to detect coimmunoprecipitated FLAG-PKS5 (lane 3) or FLAG-TTG1 (lane 4).


Figure 2. PKS5 and J3 Have Overlapping Tissue-Specific Expression and Subcellular Localization.
(A) to (J) Expression of J3 and PKS5 in 3-d-old seedlings ([A] and [F]), 10-d-old seedlings ([B] and [G]), rosette leaves ([C] and [H]), roots ([D] and [I]),and cross sections of roots ([E] and [J]).
(A) to (E) PKS5 promoter-GUS expression in wild-type seedlings.
(F) to (J) J3 promoter-GUS expression in wild-type seedlings.
(K) to (P) Subcellular localization of GFP-J3 ([K] to [M]) and PKS5-YFP ([N] to [P]) in the upper portion of the root. Confocal GFP images ([K] and [N]),bright-field images ([L] and [O]), and combined GFP and bright-field images ([M] and [P]).
(Q) Both of PKS5 and J3 were detected in soluble and plasma membrane–enriched fractions. Isolation of plasma membrane vesicles was by two-phasepartitioning from seedlings of j3-1 plants expressing 35SP:33flag-J3 or pks5-1 plants expressing DexP:33flag-PKS5. Equal amount of soluble (S) andplasma membrane (P) proteins were separated by SDS-PAGE followed by analysis with anti-flag, anti-MAPK3, or anti-PM H+-ATPase antibodies.
(R) Both of PKS5 and J3 were detected in nucleus. Nuclei were isolated from seedlings of j3-1 plants expressing 35SP:33flag-J3 or pks5-1 plantsexpressing DexP:33flag-PKS5. The nuclei pellet was then washed three times. Equal amount of three washing (S1-S3) and nuclei (N) proteins wereseparated by SDS-PAGE followed by analysis with anti-flag or anti-Histone H3 antibodies.
[See online article for color version of this figure.]


J3 Activates Plasma Membrane H+-ATPase Activity Figure 3. j3 Mutants Have Increased Sensitivity to Salt in Alkaline Conditions.
(A) Schematic diagram of the J3 gene showing the T-DNA insertion sites in the j3-1 and j3-2 mutants. The filled black boxes represent exons, while thelines between the boxes represent introns. The two T-DNA insertions are also indicated.
(B) Transcript levels of J3 are undetectable in the j3-1 and j3-2 mutants. Total RNA was isolated from 10-d-old seedlings of Col-0, j3-1, j3-2, and pks5-1mutants. RNA (15 mg) from each sample was used for RNA gel blot analysis. Ethidium bromide staining of rRNA is included as a loading control.
(C) to (O) Five-day-old Col-0, pks5-1, j3-1, j3-2, and j3-1 expressing 35SP: J3 seedlings grown on MS medium at pH 5.8 were transferred to MS mediumat pH 5.8, at pH 7.7 with 75 mM NaCl, or at pH 8.1 with 75 mM NaCl. Photographs in (C), (F), and (L) were taken 7 d after transfer; in (D), (G), and (M), 14d after transfer; in (E) and (H), 21 d after transfer.
(I) and (N) Primary root elongation of plants transferred to MS medium at pH 5.8.
(J) and (O) Primary root elongation of plants transferred to MS medium at pH 7.7 with 75 mM NaCl.
(K) Primary root elongation of plants transferred to MS medium at pH 8.1 with 75 mM NaCl.
(P) Relative expression of j3-1 expressing 35SP:J3 with J3 real-time quantitative RT-PCR.
In (I) and (N), primary root length was measured 7 d after transfer; (J) and (O), primary root length was measured 14 d after transfer; (K), primary rootlength was measured 21 d after transfer. Error bars represent SD (plant number >15). A Student's t test was used to determine statistical significance;significant differences (P # 0.05) in (I) to (K), (N), and (O) are indicated by different lowercase letters.
into the pACT2 vector, and combinations of PKS5 and J3 were with highest expression in reproductive and root tissues (see cotransformed into yeast. Both the J3C1 and J3C2 peptides Supplemental Figure 1 online).
interacted with the full-length PKS5 protein and the C terminus of To learn more about the interaction between PKS5 and J3, we PKS5, with J3C1 showing a stronger interaction than J3C2 determined the subcellular localization of the two proteins. The (Figure 1B). The PKS5 protein interacted weakly with J3 (Figure green fluorescent protein (GFP) reporter was fused to both 1B). To determine the region of PKS5 that interacts with J3, proteins at their N termini under the control of the 35S promoter, fragments encoding the N-terminal kinase domain (PKS5N, and the resulting plasmids were transformed into the Arabidopsis amino acids 1 to 281) or the C-terminal regulatory domain Col-0 genetic background. Transgenic plants in the T2 genera- (PKS5C, amino acids 282 to 435) of PKS5 were cloned into tion were tested for GFP localization using confocal microscopy.
pAS2, and these two plasmids were cotransformed with the GFP-J3 was detected at the cell membrane, in the cytoplasm, J3 plasmids into yeast. The PKS5 kinase domain (N terminus) and in the nucleus (Figures 2K to 2M); however, no GFP-PKS5 did not interact with any portion of J3 (Figure 1C). The PKS5 C signal was detected in >100 35SP:GFP-PKS5 transgenic lines.
terminus interacted with J3C1, which showed a stronger We then fused yellow fluorescent protein (YFP) to the C terminus interaction than any other J3 fragment (Figure 1D). As con- of PKS5 under the control of a dexamethasone-inducible pro- trols, PKS5 was shown to interact with SOS3-LIKE CALCIUM moter (Aoyama and Chua, 1997), and the YFP signal was BINDING PROTEIN1 (SCaBP1), and this interaction was analyzed in transgenic plants treated with 10 mM dexametha- abolished when the FISL domain (a domain in the PKS5 sone. As was found for GFP-J3, PKS5 localized to the cell protein required for SCaBP binding) was deleted (PKS5DF, membrane, in the cytoplasm, and in the nucleus (Figures 2N To determine if this interaction exists in vivo, three FLAG tags To further analyze the subcellular localization of PKS5 and J3 in a tandem repeat were fused to PKS5 or to the trichome- in plant cells, we fused a 33FLAG tag at the N terminus of PKS5 associated gene TRANSPARENT TESTA GLABRA1 (TTG1) at under the control of a dexamethasone-inducible promoter and their N termini, and six Myc tags in a tandem repeat were fused to 33FLAG tag at the N terminus of J3 driven by the 35S promoter.
J3 at its N terminus with fusions for all three genes under the The resulting plasmids and 35SP:GFP-J3 were transferred into control of the 35S promoter. Combinations of 63Myc-J3 and their corresponding mutants and the mutant phenotypes were 33FLAG-TTG1 or 63Myc-J3 and 33FLAG-PKS5 were cotrans- rescued by the transgenes (see Supplemental Figures 2 and 3 fected into Arabidopsis leaf protoplasts. The 63Myc-J3 protein online). We then isolated nuclei, a plasma membrane–enriched was immunoprecipitated using anti-Myc conjugated agarose.
fraction, and a soluble fraction from the transgenic plants After washing, immunoblots were probed with anti-FLAG anti- expressing 35SP:33FLAG-J3 and DexP:33FLAG-PKS5 and body. The 33FLAG-PKS5 but not 33FLAG-TTG1 protein was analyzed the immunoblots with anti-FLAG antibody. As shown pulled down by 63Myc-J3 (Figure 1E), suggesting that PKS5 and in Figure 2Q and 2R, the tagged PKS5 and J3 proteins were J3 can function in the same complex. Together with the yeast detected in all three fractions. These results are consistent with two-hybrid results, our data indicate that PKS5 and J3 interact in our PKS5-YFP and GFP-J3 results (Figures 2K to 2P). Using the same protein samples, as expected, MITOGEN-ACTIVATEDPROTEIN KINASE3, the PM H+-ATPase, and histone H3 werefound in the soluble, plasma membrane–enriched and nuclear PKS5 and J3 Have Overlapping Tissue-Specific Expression fractions, respectively. To further determine the purity of the phase and Subcellular Localization partitioned membrane fractions, anti-Arf1 (ADP-ribosylation To determine if PKS5 and J3 colocalize in planta, we monitored factor 1, a Golgi apparatus marker) and anti-Sar1 (secretion- PKS5 and J3 tissue specific expression using two approaches.
associated and Ras-related protein 1, an endoplasmic reticulum First, a 1918-bp DNA fragment upstream of the J3 translational marker) antibodies were used to investigate the presence of start codon was amplified and cloned into pCambia1301 tran- endoplasmic reticulum and Golgi membranes in the plasma scriptionally fused to b-glucuronidase (GUS) and the resulting membrane–enriched fraction. Both proteins were at undetect- plasmid was transformed into Columbia-0 (Col-0). GUS signals able levels in the plasma membrane–enriched fraction. Consis- driven by the PKS5 (Fuglsang et al., 2007) or J3 promoter are tent with a previous study (Pimpl et al., 2000), these proteins were shown in Figures 2A to 2E and 2F to 2J, respectively. Both J3P: detected in total membrane and soluble fractions (see Supple- GUS and PKS5P:GUS were expressed in the roots and leaves of mental Figure 4 online). Together, these data demonstrate that seedlings with stronger GUS staining in vascular tissue (Figure 2).
the gene expression and protein localization of J3 and PKS5 In cross sections of the root, PKS5P:GUS was mainly observed overlap during Arabidopsis development.
in phloem (Figure 2E), which is consistent with previous findings(Fuglsang et al., 2007), while the J3P:GUS signal was observed in j3 Mutants Have Increased Sensitivity to Salt in epidermal cells, the cortex, phloem, and xylem parenchyma cells Alkaline Conditions (Figure 2J); this expression pattern is similar to that of AHA2P:GUS (Fuglsang et al., 2007). We also analyzed the tissue-specific To determine if J3 and PKS5 have similar functions, we obtained expression of PKS5 and J3 using quantitative real-time PCR.
two J3 T-DNA insertion lines from TAIR (SALK_132923 and Total RNA was extracted from roots, stems, rosette leaves, SALK_141625). The positions of the T-DNA insertions are shown cauline leaves, flowers, and siliques of 40-d-old Col-0 plants.
in Figure 3A. Homozygous T-DNA lines, j3-1 and j3-2, were Both PKS5 and J3 were constitutively expressed in all tissues identified using T-DNA left border primers and J3 gene-specific


J3 Activates Plasma Membrane H+-ATPase Activity primers. To determine if expression of J3 is abolished in these were transferred to medium at pH 5.8, pH 7.7 with 75 mM NaCl, two lines, total RNA was extracted from 10-d-old Col-0, pks5-1 or pH 8.1 with 75 mM NaCl. No significant difference in growth (a PKS5 loss-of-function mutant; Fuglsang et al., 2007), and was detected between Col-0 and the j3 mutants on medium at j3-1 and j3-2 seedlings and analyzed using RNA gel blots. The pH 5.8 (Figures 3C and 3I; see Supplemental Figure 7A online).
expression of J3 could not be detected in j3-1 and j3-2; however, On medium at pH 7.7 with 75 mM NaCl (Figures 3D and 3J; see it is present in Col-0 and pks5-1 (Figure 3B; see Supplemental Supplemental Figure 7B online), root elongation in the j3 mutants Figure 5 online). We have previously shown that PKS5 is a was reduced compared with that of Col-0, and this reduction in negative regulator of the PM H+-ATPase and that PKS5 loss-of- growth was even more pronounced at pH 8.1 in the presence of function mutants are resistant to high pH in the external medium 75 mM NaCl (Figures 3E and 3K; see Supplemental Figure 7C (Fuglsang et al., 2007). When we monitored j3 mutant seedling online). By contrast, when we grew Col-0 and pks5-1 seedlings growth in response to alkaline pH, no consistent, significant on the same media, primary root elongation in pks5-1 seedlings difference was detected between Col-0 and mutant plants. In was less sensitive to NaCl in alkaline conditions relative to the nature, soil alkalinity is often associated with increased soil growth of wild-type plants (Figures 3G, 3H, 3J, and 3K). This salinity partly due to application of fertilizers and irrigation water result is consistent with our previous finding that pks5-1 is more (Richards, 1954). Alkaline conditions significantly increased the tolerant to alkaline pH than Col-0 (Fuglsang et al., 2007). When salt sensitivity of Arabidopsis (see Supplemental Figure 6 online).
we tested the sensitivity of the j3-1 transgenic plants expressing To determine if the j3 mutants have increased sensitivity to 35SP:J3 to salinity in alkaline conditions, we found the mutant salinity in alkaline conditions, 5-d-old seedlings of Col-0, j3-1, phenotype was rescued by the transgene (Figures 3L to 3P; see and j3-2 grown on Murashige and Skoog (MS) medium at pH 5.8 Supplemental Figures 7D and 7E online).
Figure 4. J3 Positively Regulates PM H+-ATPase Activity.
Plasma membrane vesicles were isolated from Col-0, pks5-1, j3-1, and j3-2 mutant plants treated with or without 250 mM NaCl for 3 or 6 d. PM H+-ATPase activity (H+-transport resulting in intravesicular acidification and pH gradient [DpH] formation) was initiated by addition of 3 mM ATP, and theDpH was collapsed by addition of 10 mM (final concentration) carbonyl cyanide m-chlorophenylhydrazone (CCCP).
(A) Comparison of PM H+-ATPase activity in vesicles isolated from Col-0, j3-1, j3-2, and pks5-1 plants treated with or without 250 mM NaCl for 3 or 6 d.
(B) and (C) PM H+-ATPase activity was measured in the vesicles isolated from Col-0, j3-1, j3-2, and pks5-1 plants treated with 250 mM NaCl for 3 d (B)or 6 d (C).
(D) PM H+-ATPase activity was measured at different pH.
(E) Comparison of PM H+-ATPase activity in the presence of 250 ng/mL recombinant J3 protein in vesicles isolated from Col-0, j3-1, and pks5-1 plants.
(F) PM H+-ATPase activity was measured in the presence of 250 ng/mL recombinant J3 protein in vesicles isolated from Col-0, j3-1, and pks5-1 plants.
Units of PM H+-ATPase activity (H+-transport) are change in fluorescence (DF) per min per mg protein.
All data represent means 6 SE of at least three replicate experiments. Each replicate was performed using independent membrane preparations. Onerepresentative experiment of three replicates is shown in (B) and (C). A Student's t test was used to determine statistical significance; significantdifferences (P # 0.05) in (A) and (E) are indicated by different lowercase letters.


Figure 5. Proton Efflux Decreases in the Root of j3 Mutant Plants.
The pH sensitive ratiometric probe D-1950, a dextran-conjugated membrane impermeable fluorescence dye, was used to measure proton secretion inthe upper region of the root, and MIFE assays were used for proton efflux measurements at the root apex.
(A) to (E) pH ratio imaging in the apoplast of Col-0, pks5-1, j3-1, and j3-2 roots.
(A) Fluorescence of fluorescein in pseudocolor.
(B) Fluorescence of rhodamine in pseudocolor.
(C) Overlay of the fluorescein and rhodamine fluorescence in pseudo colors. Bars = 40 mm.
(D) pH calibration curve showing the response of the probe D-1950 at different pH buffers regimes.
(E) Mean value ratio curve for mutant (n = 20) and Col-0 (n = 17) plants. Ratios were calculated as fluorescein/rhodamin fluorescence levels. The slope ineach experiment was calculated and used in one-way analysis of variance. Asterisks show where pH regimes were shifted upwards to pH 8.4 anddownwards to pH 5.8, respectively. Arrows indicate the two outer points used to define the area from where the slope was calculated.
(F) to (I) The net proton effluxes in roots of Col-0, pks5-1, j3-1, and j3-2 plants. The MIFE technique was used for noninvasive ion flux measurement.
(F) Net proton effluxes in Col-0, pks5-1, j3-1, and j3-2 root tips. After roots were treated with alkaline conditions (pH 7.7) plus 75 mM NaCl, transient netH+ fluxes were recorded.
(G) Calculated net proton effluxes from (F).
J3 Activates Plasma Membrane H+-ATPase Activity J3 Positively Regulates PM H+-ATPase Activity treatment with immunoblot analysis using PM H+-ATPase anti-bodies, we did not observe significant differences in protein Because J3 interacts with PKS5 and loss-of-function j3 and levels among Col-0, pks5-1, j3-1, and j3-2 plants (see Supple- pks5-1 mutants have opposite responses to salinity in alkaline mental Figures 10A and 10B online). Addition of J3 protein to conditions, we determined if J3 also has an effect on PM H+- membranes isolated from pks5-1 had no significant effect on ATPase activity. Plasma membrane–enriched vesicles were activity (Figures 4E and 4F). These data indicate that J3 is a novel isolated from 5-week-old Col-0, pks5-1, j3-1, and j3-2 plants regulator of PM H+-ATPase activity and that this regulation likely treated with or without 250 mM NaCl for 3 or 6 d, and the ATP takes place via mediation of PKS5 activity.
hydrolytic and H+-transport activities of the H+-ATPase weremeasured. Assays in the presence of specific H+-ATPase inhib-itors demonstrated that the vesicles were enriched in plasma Proton Efflux Is Increased in Roots of pks5-1 and Decreased membranes and that they were transport competent (see Sup- in Roots of j3 Mutants plemental Table 1 and Supplemental Figure 8 online). Unlessindicated, all PM H+-ATPase activity assays shown monitor the Previously, we have shown that the rate of proton secretion in H+-transport activity of the H+-ATPase. Without NaCl treatment, roots of pks5-1 plants is higher than that in wild type in alkaline there was no significant difference in PM H+-ATPase activity conditions (Fuglsang et al., 2007). The j3 mutants have opposite between Col-0 and the j3 mutants (Figure 4A). After plants were phenotypes in terms of PM H+ ATPase activity, sensitivity to salt, treated with NaCl, PM H+-ATPase activity increased significantly and alkalization when compared with pks5-1 plants. To deter- in all plants. However, the increase in activity was significantly mine the effect of salt and alkaline conditions on in vivo proton lower in the vesicles isolated from j3 mutant plants compared fluxes in the root of j3 seedlings, we used the pH-sensitive with activity in Col-0 (Figures 4A to 4C). Consistent with previous ratiometric probe D-1950, a dextran-conjugated membrane results (Fuglsang et al., 2007), PM H+-ATPase activity in pks5-1 impermeable fluorescence dye that reports pH changes be- mutant plants was higher than activity in Col-0 (Figures 4A to 4C).
tween pH 5.0 and pH 8, to measure proton secretion in the upper When we increased the time of treatment to 6 d, PM H+-ATPase region of the root and used microelectrode ion flux estimation activity increased further relative to activity after the 3-d treat- (MIFE) assays for proton efflux measurements at the root apex ment. The increase in Col-0 was 44%, but only 11% in pks5-1, (as described in Fuglsang et al., 2007).
14% in j3-1, and 19% in j3-2 mutants (Figures 4A and 4C). These Seven-day-old seedlings of Col-0, pks5-1, j3-1, and j3-2 results suggest that loss of either PKS5 or J3 in Arabidopsis grown on medium at pH 5.8 were preincubated with D1950 (20 alters regulation of PM H+-ATPase activity during salt stress. The mM) in a buffer containing 10 mM KCl at pH 6.0. The probe was difference in PM H+-ATPase activity between Col-0 and j3 found in the apoplast but not in the cytoplasm (Figures 5A to 5C).
increased to 32% (j3-1) and 31% (j3-2) after 6 d of treatment The seedlings were subsequently treated with KHCO3 buffer, pH compared with 14% (j3-1) and 16% (j3-2) after 3 d of treatment.
8.4, containing 75 mM NaCl. A pH increase was detected Consistent with what we have shown previously, the pH optimum immediately, and a decrease in apoplast pH in the root was for PM H+-ATPase activity in pks5-1 shifted from 6.5 to 7.0, while seen as a fluorescence change by confocal microscopy. Con- for j3 mutants, the optimum stayed at 6.5, the same as for Col-0, sistent with previous findings, the pH in the apoplast of roots in but with lower activity (Figure 4D).
the pks5-1 mutant decreased faster than in Col-0 in response to Addition of recombinant PKS5 protein to plasma membrane salt in alkaline conditions, suggesting that the pks5-1 mutant vesicles isolated from the pks5-1 mutant rescued PM H+-ATPase secretes more H+ into the apoplast. However, the rate of de- activity to Col-0 levels (Fuglsang et al., 2007). To determine if crease in the j3 mutants was largely reduced compared with addition of J3 protein in the assay also affects PM H+-ATPase Col-0, suggesting that the j3 mutant secretes less H+ into the activity, the J3 coding region was cloned into the pGEX- apoplast (Figure 5E).
6p-1 vector to generate a translational fusion to glutathione For noninvasive ion flux measurements, net H+ fluxes were S-transferase (GST). The resulting plasmid was transferred into measured in the root apex of 7-d-old seedlings of Col-0, pks5-1, E. coli BL21 (DE3), and the J3 protein was purified and added to j3-1, and j3-2. The seedlings were preincubated in buffer (0.5 mM PM H+-ATPase assays. In the presence of 250 ng/mL J3, PM H+- KCl, 0.1 mM CaCl2, and 0.3 mM MES, pH 6.0) for 20 min and ATPase activity increased in vesicles isolated from Col-0 and the assayed in the same buffer containing 75 mM NaCl at pH 7.7. The j3 mutant (Figures 4E and 4F). As a control, GST or boiled J3 transmembrane H+ efflux increased in pks5-1 and decreased in protein was added to assays and did not have any effect on PM j3 compared with Col-0 (Figures 5F and 5G). To determine if the H+-ATPase activity (see Supplemental Figure 9 online). When we changes in H+ efflux in the mutants are due to the changes in PM monitored the level of PM H+-ATPase protein in response to NaCl H+-ATPase activity, net H+ fluxes at the root apex of Col-0 and Figure 5. (continued).
(H) Vanadate treatment eliminated the net proton effluxes. The seedlings of Col-0, pks5-1, j3-1, and j3-2 were treated with alkaline conditions (pH 7.7)plus 75 mM NaCl and 1 mM vanadate and then the net proton effluxes were measured.
(I) Calculated net proton effluxes from (H). A Student's t test was used to determine statistical significance; significant differences (P # 0.05) in (G) and (I)are indicated by different lowercase letters.
[See online article for color version of this figure.]


Figure 6. PKS5 Kinase Activity Negatively Correlates with PM H+-ATPase Activity and Seedling Sensitivity to Salt in Alkaline Conditions.
(A) and (B) Kinase activity comparison for the PKS5, PKS5-3, PKS5-4, and PKS5-6 proteins. Left panel: Coomassie blue (CBB)–stained SDS-PAGE gelcontaining the PKS5 wild-type and mutant proteins and the substrate, MBP. Right panel: autoradiograph (AUD) of kinase activity assays shown in theleft panel.
(C) to (H) Five-day-old wild type (Col er105), pks5-3, pks5-4, and pks5-6 seedlings grown on MS medium at pH 5.8 were transferred to MS medium atpH 5.8, at pH 7.7 with 75 mM NaCl, or at pH 8.1 with 75 mM NaCl. Photographs in (C) and (F) were taken 7 d after transfer; in (D) and (G), 14 d aftertransfer; and in (E) and (H), 21 d after transfer.
(I) Primary root elongation of seedlings transferred to MS medium at pH 5.8.
(J) Primary root elongation of seedlings transferred to MS medium at pH 7.7 with 75 mM NaCl.
(K) Primary root elongation of seedlings transferred to MS medium at pH 8.1 with 75 mM NaCl.
In (I) to (K), primary root length was measured 7, 14, and 21 d after transfer, respectively. Error bars represent SD (plant number >15). A Student's t testwas used for determining the statistical significance; significant differences (P # 0.05) in (I) to (K) are indicated by different lowercase letters.


J3 Activates Plasma Membrane H+-ATPase Activity Figure 7. PKS5 Inhibits PM H+-ATPase Activity.
PM vesicles were isolated from wild-type (Col er105), Col-0, pks5-1 T-DNA insertion, and pks5-3, pks5-4, and pks5-6 point mutant plants treated with orwithout 250 mM NaCl for 3 d. PM H+-ATPase activity (H+-transport) was initiated by addition of 3 mM ATP, and the pH gradient was collapsed byaddition of 10 mM CCCP. PM H+-ATPase activity was measured in the vesicles as follows.
(A) Comparison of PM H+-ATPase activity in vesicles isolated from Col-0, pks5-1, wild-type, pks5-3, pks5-4, and pks5-6 plants treated with or without250 mM NaCl for 3 d.
(B) PM H+-ATPase activity was determined in the vesicles isolated from Col-0, pks5-1, wild-type, pks5-3, pks5-4, and pks5-6 plants treated with 250mM NaCl for 3 d.
(C) Comparison of PM H+-ATPase activity in vesicles isolated from pks5-1 mutant plants in the presence of 250 ng/mL PKS5, PKS5-3, PKS5-4, orPKS5-6 recombinant protein.
(D) PM H+-ATPase activity was measured in the vesicles isolated from pks5-1 mutant plants in the presence of 250 ng/mL PKS5, PKS5-3, PKS5-4, orPKS5-6 recombinant protein.
the mutants were measured in pH 7.7 buffer containing 1 mM Next we tested the NaCl sensitivity of the pks5 mutants at vanadate, an inhibitor of P-type ATPases (Figures 5H and 5I). No alkaline pH. Five-day-old wild-type, pks5-3, pks5-4, and pks5-6 difference in net H+ efflux was detected for Col-0, pks5-1, j3-1, or seedlings grown on medium at pH 5.8 were transferred to j3-2, and vanadate eliminated H+ extrusion in all plants tested.
medium at pH 5.8, pH 7.7 with 75 mM NaCl, or pH 8.1 with 75 Taken together, our results suggest that PM H+-ATPase activity mM NaCl. No significant growth differences were detected is a major factor contributing to the higher rate of proton between the wild type and pks5 mutants on medium at pH 5.8 secretion in the pks5-1 root and the lower rate in j3 mutants in (Figures 6C, 6F, and 6I; see Supplemental Figure 13A online). On salt and alkaline conditions.
medium at pH 7.7 with NaCl, root growth of pks5-3 and pks5-4(which had proteins with higher kinase activity than the wild-typePKS5 protein) was significantly reduced compared with the wild PKS5 Activity Negatively Correlates with PM H+-ATPase type (Figures 6D and 6J; see Supplemental Figure 13B online).
Activity and Seedling Sensitivity to Salt in Root elongation in the pks5-3 and pks5-4 mutants was reduced Alkaline Conditions compared with that in wild type, and this reduction in growth was To further demonstrate that J3 regulates PM H+-ATPase activity even more pronounced at pH 8.1 in the presence of 75 mM NaCl by mediating PKS5 kinase activity, we isolated pks5 mutants with (Figures 6E and 6K; see Supplemental Figure 13C online).
differing levels of kinase activity. To do this, we screened a tilling However, the root growth of pks5-6 (which had protein with (targeting-induced local lesions in genomes) mutant pool (Till much lower kinase activity than the wild-type PKS5 protein) was et al., 2003) and isolated three pks5 point mutation alleles. Tilling significantly greater than the growth of the wild type (Figures 6G, mutants were generated in the Col er105 genetic background 6H, 6J, and 6K). These results demonstrate that PKS5 activity (Torii et al., 1996), hereafter referred to as the wild type. We negatively correlates with root growth on media with salt at backcrossed the mutants three times into Col-0. These muta- alkaline pH.
tions are distributed throughout the protein, including in the Our previous data suggested that PKS5 is a negative regulator N-terminal kinase domain (pks5-4) and in the C-terminal regula- of the PM H+-ATPase (Fuglsang et al., 2007). To further demon- tory domain (pks5-3) (see Supplemental Figure 11 online). In the strate the link between PKS5 kinase and PM H+-ATPase activ- pks5-3 mutant, the Ser at position 317 in the FISL motif was ities, plasma membrane vesicles were isolated from leaves of mutated to Leu; in the pks5-4 mutant, the Ala at position 168 in Col-0, wild-type, pks5-1, pks5-3, pks5-4, and pks5-6 plants the kinase activation loop was mutated to Val; while in the pks5-6 treated with or without 250 mM NaCl, and PM H+-ATPase activity mutant, the Gly at position 219 in the kinase domain was mutated was measured. Without NaCl treatment, Col-0, the wild type, and to Ser (see Supplemental Figure 11 online). Both the kinase the mutants had similar levels of PM H+-ATPase activity, and salt activation loop and the FISL motif have been shown to be stress increased their activity but to different levels (Figures 7A important for PKS activity (Guo et al., 2001). We first tested and 7B). Consistent with previous observations, PM H+-ATPase whether these mutations affect PKS5 activity. PKS5 cDNAs were activity (quenching of fluorescence) was much higher in vesicles amplified from Col-0 and pks5 mutants and cloned into the isolated from pks5-1 than in vesicles isolated from Col-0 (Figures pQE30 vector containing a HIS tag. The fusion proteins were 7A and 7B). Similar results were observed with vesicles isolated purified from E. coli using His affinity chromatography, and the from the pks5-6 mutant. PM H+-ATPase activity in vesicles purified PKS5 proteins were used in kinase assays. PKS5 has isolated from the pks5-3 and pks5-4 mutants was much lower been shown to be an active kinase in both auto- and trans- than that of the wild type (Figures 7A and 7B). These results (substrate) phosphorylation (Fuglsang et al., 2007). When provide further support for a negative correlation between PM compared with the activity of the wild-type PKS5 protein, re- H+-ATPase activity and PKS5 kinase activity.
combinant PKS5-3 and PKS5-4 proteins were more active; To provide additional evidence that changes in PM H+-ATPase however, PKS5-6 was less active in both autophosphorylation activity in the pks5 mutants are due to changes in PKS5 kinase and Myelin Basic Protein (MBP) phosphorylation (Figures 6A and activity, we added recombinant PKS5 proteins to transport 6B; see Supplemental Figure 12 online).
assays with plasma membrane vesicles isolated from the pks5-1 Figure 7. (continued).
(E) Comparison of PM H+-ATPase activity in vesicles isolated from wild-type plants in the presence of 250 ng/mL PKS5, PKS5-3, PKS5-4, or PKS5-6recombinant protein.
(F) PM H+-ATPase activity was measured in vesicles isolated from wild-type plants in the presence of 250 ng/mL PKS5, PKS5-3, PKS5-4, or PKS5-6recombinant protein.
(G) Comparison of PM H+-ATPase activity in vesicles isolated from pks5-6 mutant plants in the presence of 250 ng/mL of recombinant PKS5-6 protein.
(H) The PM H+-ATPase activity was measured in the vesicles isolated from pks5-6 mutant plants in the presence of 250 ng/mL PKS5-6 recombinantprotein.
The units of the PM H+-ATPase activity are DF/min per mg protein. All data represent means 6 SE of at least three replicate experiments. Each replicatewas performed using independent membrane preparations. One representative experiment of three replicates is shown in (B), (D), (F), and (H). AStudent's t test was used to determine statistical significance; significant differences (P # 0.05) in (A), (C), (E), and (G) are indicated by differentlowercase letters.


J3 Activates Plasma Membrane H+-ATPase Activity mutant. Consistent with previous studies, wild-type PKS5 pro-tein significantly reduced PM H+-ATPase activity in vesiclesisolated from the pks5-1 mutant (Figures 7C and 7D) and had noeffect on the PM H+-ATPase activity of vesicles isolated fromCol-0 plants (Figures 7E and 7F). Recombinant PKS5-6 proteinhad no effect on PM H+-ATPase activity in the vesicles isolatedfrom Col-0 or the pks5-1 and pks5-6 mutants (Figures 7C to 7H).
When either PKS5-3 or PKS5-4 protein was added to vesiclesisolated from Col-0 or the pks5-1 mutant, PM H+-ATPase activitywas reduced; however, this effect was much more dramatic inpks5-1 compared with Col-0 (Figures 7C to 7F). When used ascontrols, boiled recombinant PKS recombinant proteins did nothave any effect on PM H+-ATPase activity. These results supportthe conclusion that PKS5 kinase activity is negatively correlatedwith PM H+-ATPase activity.
J3 Functions Upstream of PKS5 in the Regulation of PMH+-ATPase Activity To determine whether PKS5 genetically interacts with J3, wecrossed j3-1 to pks5-1, pks5-3, or pks5-4 to generate j3-1 pks5-1, j3-1 pks5-3, and j3-1 pks5-4 double mutants. T-DNA insertionsin pks5-1 and j3-1 were confirmed using gene-specific primers,and the pks5-3 and pks5-4 mutations were confirmed usingderived cleaved amplified polymorphic sequences (dCAPS)primer-based PCR followed by sequencing of the mutations.
To assay PM H+-ATPase activity, plasma membrane–enrichedvesicles were isolated from Col-0 and double mutant plants Figure 8. J3 Regulates PM H+-ATPase Activity through PKS5.
treated with or without 250 mM NaCl. As shown in Figure 8, the Plasma membrane vesicles were isolated from Col-0, j3-1 pks5-1, j3-1 PM H+-ATPase activity of the salt-treated j3-1 pks5-1 double pks5-3, and j3-1 pks5-4 mutant plants treated with or without 250 mM mutant was similar to the activity of the pks5-1 single mutant, and NaCl for 3 d. PM H+-ATPase activity was initiated by addition of 3 mM the activities of both were higher than the activity in Col-0 after ATP, and the pH gradient was collapsed by addition of 10 mM CCCP. PM salt treatment. These results indicate that, genetically, J3 func- H+-ATPase activity was measured in the vesicles as follows.
tions upstream of PKS5. Furthermore, PM H+-ATPase activity in (A) Comparison of PM H+-ATPase activity in vesicles isolated from Col-0, both the j3-1 pks5-3 and j3-1 pks5-4 double mutants was similar j3-1, pks5-3, j3-1 pks5-3, pks5-4, j3-1 pks5-4, pks5-1, and j3-1 pks5-1plants treated with or without 250 mM NaCl for 3 d.
to the activity of their respective pks5 parent and lower than that (B) PM H+-ATPase activity was determined in vesicles isolated from of the j3-1 parent (Figure 8). These results demonstrate that J3 Col-0, j3-1 pks5-1, j3-1 pks5-3, and j3-1 pks5-4 plants treated with 250 regulates PM H+-ATPase activity by mediating PKS5 kinase mM NaCl for 3 d.
The units of PM H+-ATPase activity are DF/min per mg protein. All data To determine if the altered PM H+-ATPase activity in the dou- represent means 6 SE of at least three replicate experiments. Each ble mutants correlates with seedling responses to salt in alkaline replicate was performed using independent membrane preparations.
conditions, 5-d-old j3-1, pks5-1, j3-1 pks5-1, pks5-3, j3-1 pks5-3, One representative experiment of three replicates is shown in (B). A pks5-4, and j3-1 pks5-4 plants grown on medium at pH 5.8 were Student's t test was used to determine statistical significance; significant transferred to MS medium at pH 5.8, pH 7.7 with 75 mM NaCl, differences (P # 0.05) in (A) are indicated by different lowercase letters.
or pH 8.1 with 75 mM NaCl. Consistent with measurements ofPM H+-ATPase activity, all of the double mutants showedphenotypes similar to their pks5 parent (Figure 9; see Supple- this hypothesis, a protein kinase assay was performed. As mental Figure 14 online), suggesting that J3 regulates PM predicted, J3 repressed PKS5 kinase activity (Figures 10A to H+-ATPase activity and plant response to salt at alkaline pH by 10C and 10E), and the more J3 protein that was added to the mediating PKS5 activity.
reaction, the more PKS5 activity was inhibited. The specificity ofthis repression was shown based on the lack of J3 repression ofthe kinase activity of SOS2 (Figures 10D and 10E), a PKS5 J3 Represses PKS5 Kinase Activity Our results demonstrate that J3 interacts with and functions When we overexpressed J3 in the pks5-1 and pks5-3 mutants, genetically upstream of PKS5. In addition, these proteins have the pks5-3 salt sensitive phenotype in alkaline conditions was opposite effects on the regulation of PM H+-ATPase activity and rescued, whereas the phenotype of pks5-1 was not significantly seedling sensitivity to salt at alkaline pH. One explanation for altered (Figures 10F to 10S; see Supplemental Figure 15 online).
these observations is J3 represses PKS5 kinase activity. To test These results further support the conclusion that J3 regulation of Figure 9. The j3-1 pks5 Double Mutants Phenocopy the pks5 Response to Salt in Alkaline Conditions.
(A) to (I) Five-day-old j3-1, pks5-1, j3-1 pks5-1, pks5-3, j3-1 pks5-3, pks5-4, and j3-1 pks5-4 mutant seedlings grown on MS medium at pH 5.8, at pH 7.7with 75 mM NaCl, or at pH 8.1 with 75 mM NaCl. Photographs in (A), (D), and (G) were taken 7 d after transfer; in (B), (E), and (H), 14 d after transfer; andin (C), (F), and (I), 21 d after transfer.
(J) Primary root elongation of plants transferred to MS medium at pH 5.8.
(K) Primary root elongation of plants transferred to MS medium at pH 7.7 with 75 mM NaCl.
(L) Primary root elongation of plants transferred to MS medium at pH 8.1 with 75 mM NaCl.
In (J) to (L), primary root length was measured 7, 14, and 21 d after transfer, respectively. Error bars represent SD (plant number >15). A Student's t testwas used to determine statistical significance; significant differences (P # 0.05) in (J) to (L) are indicated by different lowercase letters.
J3 Activates Plasma Membrane H+-ATPase Activity Figure 10. J3 Represses PKS5 Kinase Activity.
Increasing concentrations of J3 protein were incubated in kinase assay buffer with PKS5 (A), PKS5-3 (B), PKS5-4 (C), or SOS2 (D) protein. Top panel:SDS-PAGE gel with MBP or GST-P3 stained with Coomassie blue (CBB). Lower panel: MBP or GST-P3 (P3; a synthetic peptide designed based on therecognition sequences of protein kinase C or SNF1/AMPK kinases, ALARAASAAALARRR) substrate phosphorylation (AUD).
(E) Quantification of data shown in (A) to (D).
(F) and (G) Overexpression of J3 in pks5-1 and pks5-3 mutants. Total RNA was extracted from 12-d-old seedlings of pks5-1, pks5-3, and the response of the plant to salt in alkaline conditions takes place activity (Figures 8 to 10). Interestingly, j3-1 pks5-3 and j3-1 pks5-4 via repression of PKS5 kinase activity.
double mutants have similar levels of PM H+-ATPase activity andsimilar sensitivity to growth in media with NaCl at alkaline pHto what is seen for their pks5 parent (Figures 8 and 9). The fact that the phenotypes in these double mutants are not more severesuggests that other, as yet unidentified, components may also be The PM H+-ATPase is a highly regulated enzyme with numerous involved in the regulation of phosphorylation/dephosphorylation physiological functions (Duby et al., 2009). Evidence exists for of Ser-931 and that there is a threshold effect of PKS5 kinase changes in phosphorylation status of the H+-ATPase leading to activity on the regulation of PM H+-ATPase activity.
either activation or inhibition of enzyme activity (Vera-Estrella PM H+-ATPase activity is stimulated by many environmental et al., 1994; Xing et al., 1996; Schaller and Oecking, 1999).
changes, known to be regulated by a calcium-dependent Activation of the enzyme requires phosphorylation of its C SCaBP1-PKS5 pathway. When the Arabidopsis proteins are terminus leading to the binding of 14-3-3 proteins and the expressed in yeast, repression of PM H+-ATPase activity by removal of an autoinhibition by the R domain. Several phosphor- PKS5 is dependent on the presence of SCaBP1 (Fuglsang et al., ylation sites have been identified in the C-terminal region of the 2007); however, it is currently not known how SCaBP1 regulates protein (Duby et al., 2009). While little is known about the protein the PKS5 protein. The predicted functions of the SCaBP proteins kinases or phosphatases that are directly responsible for altering are to perceive changes in intracellular calcium levels (calcium PM H+-ATPase phosphorylation status, several proteins have sensors) and then to interact with, activate, and recruit PKS been implicated in the C-terminal phosphorylation events lead- kinase proteins to cell membranes to activate their targets ing to binding of the regulatory 14-3-3 protein (Xing et al., 1996; (Halfter et al., 2000; Quintero et al., 2002; Xu et al., 2006; Lino et al., 1998; Svennelid et al., 1999; Fuglsang et al., 2006).
Fuglsang et al., 2007; Quan et al., 2007; Lin et al., 2009). It has PKS5 was identified as a protein that negatively regulates PM been shown that ANJ1, a DnaJ-like protein in Atriplex nummu- H+-ATPase activity and controls intracellular pH homeostasis in laria, is farnesylated and geranylgeranylated in planta (Zhu et al., response to alkaline pH (Fuglsang et al., 2007). PKS5 phosphor- 1993). These modifications rely on a Cys in the CAQQ motif at the ylates Ser-931 on the AHA2 isoform of the PM H+-ATPase, C terminus of the ANJ1 protein and increase the association of which, in turn, blocks interaction between AHA2 and an activat- ANJ1 with the cell membrane (Zhu et al., 1993). This CAQQ motif ing 14-3-3 protein. Phosphorylation of this residue is indepen- is absolutely conserved in the J3 C terminus, suggesting that, dent of Thr-947 phosphorylation of AHA2 (Fuglsang et al., 2007).
during growth in NaCl at alkaline pH, J3 is prenylated and this In the tobacco PMA2 isoform, the corresponding residue (Ser- leads to the localization of J3 to the plasma membrane and to 938) is phosphorylated in vivo (Duby et al., 2009), although it is activation of PM H+-ATPase via inhibition of PKS5 or, alterna- not known whether a SOS2-like protein kinase is involved in this tively, by modifying the affinity of PKS5 for the H+-ATPase.
In summary, we identified a new component of the PKS5 signal In this study, we identified a component in the PKS5 signaling transduction pathway that positively regulates PM H+-ATPase pathway. J3 shares similar patterns of tissue-specific expression activity in Arabidopsis. Our work provides additional evidence and subcellular localization with PKS5 and interacts with PKS5 in that regulation of the phosphorylation status of the PM H+- planta (Figures 1 and 2). However, j3 knockout mutants display ATPase is critical for the response of Arabidopsis to environ- the opposite phenotype from pks5 loss-of-function mutants, with mental stimuli.
the j3 mutants displaying increased sensitivity to NaCl at alkalinepH and decreased PM H+-ATPase activity (Figures 3 and 4).
Double mutant analysis suggests that J3 relies on and functions upstream of PKS5. Overexpression of J3 rescues the pks5-3 (aPKS5 constitutively active mutant) salt-sensitive phenotype in alkaline conditions but does not alter pks5-1 (a PKS5 T-DNA Plants were grown in controlled growth chambers under a 16-h-light knockout mutant) phenotype (Figure 10). These findings are (228C)/8-h-dark (208C) cycle. The following Arabidopsis thaliana strains consistent with the observation that J3 represses PKS5 kinase were used in this study: Col-0 and Col erecta105 (wild type), which was Figure 10. (continued).
overexpression J3 transgenic plants in both the pks5-1 and pks5-3 background. The resulting cDNAs were used for real-time PCR analysis. Error barsindicate SD (n = 3).
(H) to (O) Five-day-old pks5-1, pks5-3, wild-type, Col-0 seedlings, and seedlings overexpressing J3 in pks5-1 and pks5-3 grown on MS medium at pH5.8 were transferred to MS medium at pH 5.8 or at pH 7.7 with 75 mM NaCl. Photographs in (H), (J), (L), and (N) were taken 7 d after transfer; in (I), (K),(M), and (O), 14 d after transfer.
(P) and (R) Primary root elongation of seedlings transferred to MS medium at pH 5.8.
(Q) and (S) Primary root elongation of seedlings transferred to MS medium at pH 7.7 with 75 mM NaCl.
Primary root length was measured 7 d after transfer in (H), (J), (L), and (N) and 14 d after transfer in (I), (K), (M), and (O). Error bars represent SD (plantnumber >15). A Student's t test was used for determining the statistical significance; significant differences (P # 0.05) in (P) to (S) are indicated bydifferent lowercase letters.
J3 Activates Plasma Membrane H+-ATPase Activity used for generating Tilling mutants. For salt treatment, 5-week-old plants RNA Gel Blot Analysis of j3 Null Alleles were treated with or without 250 mM NaCl for 3 or 6 d. The Tilling mutants Total RNA was isolated from 10-d-old seedlings of Col-0, pks5-1, j3-1, (Till et al., 2003) pks5-3, pks5-4, and pks5-6 were obtained from the ABRC and j3-2 mutants. RNA (15 mg) from each sample was used for RNA gel (Alonso et al., 2003), and homozygous lines were identified by dCAPS- blot analysis (Guo et al., 2001).
based PCR and sequencing. The following dCAPS primer sequences wereused: pks5-3, 59-GCGTTTGATTTGATTTCTTACTCCT-39 and 59-CACCA-CAAGCAAATCATTCAA-39 were used to amplify a 263-bp DNA fragment Confocal Images and Proton Efflux Measurements with an AvaI site to distinguish pks5-3 and the wild type; pks5-4, GFP-J3 was constructed by excising the J3 open reading frame from a His-J3 plasmid with BamHI and SalI and cloning it into the pCAMBIA1205- TTC-39 harboring an MluI site were used to amplify a 236-bp DNA GFP binary vector downstream of GFP. The coding sequences of PKS5, fragment; pks5-6, 59-GTCTTGTTCGTTCTCGTCACC-39 and 59-CTGATC- PKS5-3, PKS5-4, and PKS5-6 were introduced into the pTA7002 vector TTCGATCTCGTCATC-39 harboring an AgeI site were used to amplify a after PCR amplification with the primers 59-CCGCTCGAGATGCCA- 252-bp fragment. j3 T-DNA insertion mutants (SALK_132923 and SALK_141625) were obtained from ABRC and identified using the following GAC-39, which introduced XhoI restriction sites at each end. These primer sequences: 59-GCTGTTGACGGCTTAGGTAG-39 or 59-TTCGAC- constructs were transformed into Col-0 plants. Five-day-old transgenic TCGATCTTGCGTTT-39 and left border T-DNA primers LBa1 59-TGGTT- T2 GFP-J3 lines were used for subcellular localization. Five-day-old T2 CACGTAGTGGGCCATCG-39 and LBb1 59-GCGTGGACCGCTTGCTG- PTA7002-PKS5-YFP transgenic plants were treated with 10 mM dexa- CAACT-39. j3-1 pks5-1, j3-1 pks5-3, and j3-1 pks5-4 double mutants methasone (Sigma-Aldrich) before analysis. GFP-J3 and PKS5-YFP fluo- were obtained by crossing j3-1 to pks5-1, pks5-3, and pks5-4 respectively, rescence was detected and images collected on a Zeiss 510 META and confirmed by PCR and sequencing.
confocal microscope. Proton efflux measurements and pH ratio imaging inthe apoplast were as described (Fuglsang et al., 2007).
Plasmid Construction The full-length PKS5 coding sequence was amplified by RT-PCR using Promoter-GUS Analysis A 1918-bp DNA fragment from 28 to 21926 bp upstream of the transla- TAAATAGCCGDGTTTGTTG-39 primers harboring a BamHI or EcoRI site, tional start site of J3 was amplified with primers 59-AGCGTCGACTGATT- respectively. The PCR products were cloned into the pGEX-6p-1 vector between BamHI and EcoRI sites. The coding sequences of PKS5-3, ACCCTA-39 and cloned into the NcoI and SalI sites of the pCAMBIA1301 PKS5-4, and PKS5-6 were also cloned into pGEX-6p-1. All fragments vector. The plasmid was transformed into wild-type Arabidopsis, and containing PKS5 and mutant coding sequences were digested from transgenic T2 lines were used for GUS assays.
pGEX-6p-1 with BamHI and SalI and subcloned into pQE30 to generatefusions to a His-tag. J3 was amplified using primers 59-CGGGATC-CATGTTCGGTAGAGGACCCTC-39 and 59-GCGTCGACTTACTGCTGG- Quantitative Real-Time PCR GCACATTGCA-39 and cloned into the BamHI and SalI sites of pET28a. J3 Total RNA was extracted with the TRI reagent (Ambion) from 10-d-old was then subcloned from this plasmid into other expression vectors as seedlings. Ten micrograms of total RNA was treated with RNase-free indicated. All plasmids were verified by sequencing.
DNase I (Promega) to remove DNA, and 2 mg of treated RNA was used forreverse transcription with M-MLV Reverse transcriptase (Promega) Kinase Activity Assays according to the manufacturer's protocol. Real-time PCR was performed Fusion constructs were transformed into Escherichia coli strain BL21 using an ABI 7500 Fast Real-Time PCR instrument and SYBR Premix (DE3). The recombinant proteins were purified with glutathione-Sepharose Ex Taq kit (TaKaRa). Gene expression levels were standardized using (Amersham Pharmacia) according to the manufacturer's protocol. Kinase ACTIN2 as an internal control. The following primer sequences were activity assays were started by mixing 100 ng of the kinase and 500 ng of used: for PKS5 expression, forward 59-TCGTCGGGATTGGATTTGTC-39 MBP in 20-mL reactions with kinase assay buffer (20 mM Tris-HCl, pH 7.2, and reverse 59-TCCATCTCAAACCCATACTC-39; for J3 expression, for- ward 59-TTCTACCTAAGCCGTCAAC-39 and reverse 59-CGTCATCAT- 2, 0.5 mM CaCl2, 10 mM ATP, and 2 mM DTT). After addition of 0.3 mL of [g-32P]ATP, the mixture was incubated at 308C for 30 min. The CATAAGCCTC-39; for ACTIN2, forward 59-GTCGTACAACCGGTA- reaction was terminated by the addition of 4 mL of 53 SDS loading buffer, TTGTG-39 and reverse 59-GAGCTGGTCTTTGAGGTTTC-39.
and the products were separated on 12% SDS-PAGE gels. The gels werestained with Coomassie Brilliant Blue, exposed on a storage phosphor screen, and phosphorylation visualized using a Typhoon 9410 phosphor The plasmids were purified using a Plasmid Maxprep Kit (Vigorous imager (Amersham).
Biotechnology) and introduced into Arabidopsis mesophyll protoplasts(Sheen, 2001). Coimmunoprecipitation was performed as described Yeast Two-Hybrid Assays (Quan et al., 2007).
The PKS5 coding sequence was cloned into the pAS2 vector between theNcoI and SalI sites. PKS5N, encoding the N-terminal 281amino acids of Plasma Membrane and Nuclei Isolation PKS5, and PKS5C, encoding the C-terminal 154 amino acids, wereamplified by PCR and were also cloned between the NcoI and SalI sites of Plasma membrane–enriched vesicles were isolated from 5-week-old pAS2. The J3 coding sequence was cloned into the pACT2 vector plants using aqueous two-phase partitioning as described (Qiu et al., between the BamHI and XhoI sites. Plasmid DNA (50 mg total) from an 2002). All steps were performed at 48C or on ice. Plants were homoge- Arabidopsis cDNA library (ABRC) was transformed into the yeast Y190 nized in isolation buffer containing 0.33 M sucrose, 10% (w/v) glycerol, strain harboring the pAS2-PKS5 plasmid. Yeast transformation and 0.2% (w/v) BSA, 5 mM EDTA, 5 mM DTT, 5 mM ascorbate, 0.2% (w/v) growth assays were performed as described in the Yeast Protocols casein, 0.6% (w/v) polyvinylpyrrolidone, 1 mM PMSF, 13 protease Handbook (Clontech). Two positive clones were identified, and sequence inhibitor, and 50 mM HEPES-KOH, pH 7.5. Two to four milliliters of analysis revealed that they are identical to At3g44110 (J3).
homogenization buffer were used per gram of tissue. The homogenate was filtered through two layers of Miracloth and centrifuged at 13,000g for Supplemental Figure 3. Phenotypic Complementation of pks5-1 by 10 min. The supernatant then was centrifuged for 50 min at 80,000g to Expression of DexP:33flag-PKS5.
obtain a microsomal pellet that was resuspended in a buffer containing Supplemental Figure 4. PKS5 and J3 Were Detected in Soluble and 0.33 M sucrose, 3 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 13 Plasma Membrane–Enriched Fractions.
protease inhibitor, and 5 mM potassium phosphate, pH 7.8. The sus-pension was added to a phase mixture to obtain a phase system Supplemental Figure 5. RT-PCR Analysis of J3 in Col-0, j3-1, and consisting of 6.2% (w/w) Dextran T-500 and 6.2% (w/w) polyethylene j3-2 Plants Using ACTIN2 as Control.
glycol 3350 in 5 mM potassium phosphate, pH 7.8, buffer containing Supplemental Figure 6. Alkaline Conditions Significantly Enhance 0.33 M sucrose and 3 mM KCl. The final upper phases were collected, Arabidopsis Sensitivity to Salt.
diluted with resuspension buffer containing 0.33 M sucrose, 10% (w/v) Supplemental Figure 7. Fresh Weight of Col-0, pks5-1, j3-1, and j3-2 glycerol, 0.1% (w/v) BSA, 0.1 mM EDTA, 2 mM DTT, 13 protease under Salt and Alkalinity Conditions.
inhibitor, and 20 mM HEPES-KOH, pH 7.5, and centrifuged for 50 minat 100,000g. The resulting pellet was collected and resuspended with Supplemental Figure 8. Vesicles Isolated from Arabidopsis Leaves resuspension buffer containing 1 mM EDTA. Isolation of a nuclei-enriched Are Transport Competent.
fraction was performed as described (Bowler et al., 2004).
Supplemental Figure 9. GST Protein Has No Effect on PM H+-ATPase Activity.
PM H+-ATPase Activity Assays Supplemental Figure 10. Immunoblot of PM H+-ATPase Protein fromCol-0, pks5-1, j3-1, and j3-2 Plants.
To characterize the activity of PM H+-ATPase, its H+-transport activitywas measured as described (Qiu et al., 2002) using 50 mg of plasma Supplemental Figure 11. Schematic Diagram of Domains and pks5 membrane protein. When used, recombinant protein (250 ng/mL) was Point Mutations' Distribution in the PKS5 Protein.
preincubated for 10 min at room temperature with plasma membrane Supplemental Figure 12. Kinase Assay for Recommbinant Proteins vesicles. An inside-acid pH gradient (DpH) was formed in the vesicles by PKS5, PKS5-3, and pET28a Empty Vector.
the activity of the H+-ATPase and measured as a decrease (quench) in the Supplemental Figure 13. Fresh Weight of the Wild Type, pks5-3, fluorescence of quinacrine (a pH-sensitive fluorescent probe). Assays (2 pks5-4, and pks5-6 under Salt and Alkalinity Conditions.
mL) contained 5 mM quinacrine, 3 mM MgSO4, 100 mM KCl, 25 mM 1,3-bis[Tris(hydroxylmethyl)methylamino]propane-HEPES, pH 6.5, 250 mM Supplemental Figure 14. Fresh Weight of Col-0, j3-1, pks5-1, j3-1 mannitol, and 50 mg/mL of plasma membrane protein. Reactions were pks5-1, pks5-3, j3-1 pks5-3, pks5-4, and j3-1 pks5-4 Seedlings under mixed by inversion several times and then placed in a dark chamber in a Salt and Alkalinity Conditions.
fluorescence spectrophotometer (Hitachi F-4500). Reactions were equil- Supplemental Figure 15. Fresh Weight of j3-1, pks5-1, pks5-3, and ibrated in the dark with stirring for 5 min before beginning fluorescence pks5-1 (pks5-1:J3) or pks5-3 Overexpressing J3 (pks5-3:J3) under readings. The assay was initiated by the addition of ATP to a final Salt and Alkalinity Conditions.
concentration of 3 mM, and formation of DpH was measured at excitationand emission wavelengths of 430 and 500 nm, respectively. At the end Supplemental Table 1. Vesicles Isolated from Arabidopsis Leaves of each reaction, 10 mM (final concentration) of the protonophore, Are Enriched in Plasma Membranes.
m-chlorophenylhydrazone (CCCP) was added to dissipate any remainingpH gradient. Specific activity was calculated by dividing the change influorescence by the mass of plasma membrane protein in the reaction perunit time (DF/min per mg of protein). Unless indicated, all data represent means 6 SE of at least three replicate experiments.
We thank Shidi Li and Jun Zhang for excellent technical assistance,Juan Sun and Lihua Hao from Xuyue (Beijing) Science and Technologyfor the MIFE assay, and the ABRC (Ohio State University) for the T-DNA Determination of Plasma Membrane Purity insertion and Tilling lines. This work was supported by the National To characterize the membrane origin and purity of the membrane ves- Basic Research Program of China (Grant 2006CB100100), the National icles, H+-ATPase substrate hydrolytic activity was determined by mea- High Technology Research and Development Program of China 863 suring the release of Pi from ATP in the presence or absence of (Grant 2008AA022304 to Y.G.), and U.S. Department of Energy/Energy H+-ATPase specific inhibitors (Yan et al., 2002).
Biosciences (Grant DE-FG02-04ER15616 to K.S.S.).
Accession Numbers Received June 25, 2009; revised March 16, 2010; accepted March 30, Sequence data from this article can be found in the Arabidopsis Genome 2010; published April 23, 2010.
Initiative under the following accession numbers: PKS5, At2g30360; J3,At3g44110.
Supplemental Data Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of The following materials are available in the online version of this article.
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Chaperone J3 Regulates the Plasma Membrane H
-ATPase through Interaction
with the PKS5 Kinase
Yongqing Yang, Yunxia Qin, Changgen Xie, Feiyi Zhao, Jinfeng Zhao, Dafa Liu, Shouyi Chen, Anja T.
Fuglsang, Michael G. Palmgren, Karen S. Schumaker, Xing Wang Deng and Yan Guo PLANT CELL published online Apr 23, 2010; DOI: 10.1105/tpc.109.069609 This information is current as of April 25, 2010 THE PLANT CELL Sign up for eTOCs for CiteTrack Alerts
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NGUYEN THI NGOC PHUONG PROFESSOR TREATMENT OF MENOPAUSE: HORMONAL and NON-HORMONAL THERAPY Prof. NGUYEN THI NGOC PHUONG, MD. Vice President of Vietnam Asociation of Gynecologists and Obstetricians President of Ho Chi Minh City Society for Reproductive Medicine THE FRANCE - VIETNAM – ASIA PACIFIC CONFERENCE