An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast–xylem interface
2007; Springer Nature; Volume: 26; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601757
ISSN1460-2075
AutoresSantiago Alejandro, Pedro L. Rodrı́guez, José Marı́a Bellés, Lynne Yenush, María Jesús García‐Sánchez, José A. Fernández, Ramón Serrano,
Tópico(s)Plant nutrient uptake and metabolism
ResumoArticle14 June 2007free access An Arabidopsis quiescin-sulfhydryl oxidase regulates cation homeostasis at the root symplast–xylem interface Santiago Alejandro Santiago Alejandro Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Pedro L Rodríguez Pedro L Rodríguez Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Jose M Bellés Jose M Bellés Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Lynne Yenush Lynne Yenush Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author María J García-Sanchez María J García-Sanchez Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, Málaga, Spain Search for more papers by this author José A Fernández José A Fernández Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, Málaga, Spain Search for more papers by this author Ramón Serrano Corresponding Author Ramón Serrano Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Santiago Alejandro Santiago Alejandro Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Pedro L Rodríguez Pedro L Rodríguez Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Jose M Bellés Jose M Bellés Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Lynne Yenush Lynne Yenush Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author María J García-Sanchez María J García-Sanchez Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, Málaga, Spain Search for more papers by this author José A Fernández José A Fernández Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, Málaga, Spain Search for more papers by this author Ramón Serrano Corresponding Author Ramón Serrano Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain Search for more papers by this author Author Information Santiago Alejandro1, Pedro L Rodríguez1, Jose M Bellés1, Lynne Yenush1, María J García-Sanchez2, José A Fernández2 and Ramón Serrano 1 1Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia-CSIC, Camino de Vera s/n, Valencia, Spain 2Departamento de Biología Vegetal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, Málaga, Spain *Corresponding author. Instituto de Biología Molecular y Celular de Plantas, Universidad Politecnica de Valencia-CSIC, Camino de Vera s/n, Valencia 46022, Spain. Tel.: +34 96 387 7883; Fax: +34 96 387 7859; E-mail: [email protected] The EMBO Journal (2007)26:3203-3215https://doi.org/10.1038/sj.emboj.7601757 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A genetic screen of Arabidopsis 'activation-tagging' mutant collection based on tolerance to norspermidine resulted in a dominant mutant (par1-1D) with increased expression of the QSO2 gene (At1g15020), encoding a member of the quiescin-sulfhydryl oxidase (QSO) family. The par1-1D mutant and transgenic plants overexpressing QSO2 cDNA grow better than wild-type Arabidopsis in media with toxic cations (polyamines, Li+ and Na+) or reduced K+ concentrations. This correlates with a decrease in the accumulation of toxic cations and an increase in the accumulation of K+ in xylem sap and shoots. Conversely, three independent loss-of-function mutants of QSO2 exhibit phenotypes opposite to those of par1-1D. QSO2 is mostly expressed in roots and is upregulated by K+ starvation. A QSO2∷GFP fusion ectopically expressed in leaf epidermis localized at the cell wall. The recombinant QSO2 protein, produced in yeast in secreted form, exhibits disulfhydryl oxidase activity. A plausible mechanism of QSO2 action consists on the activation of root systems loading K+ into xylem, but different from the SKOR channel, which is not required for QSO2 action. These results uncover QSOs as novel regulators of ion homeostasis. Introduction The homeostasis of monovalent cations is a fundamental activity of living cells, with both permissive and regulatory roles in many cellular functions. The transport of K+, Na+ and H+ determines membrane potential, turgor, intracellular cation concentrations and pH, and these basic parameters are crucial for nutrient uptake, metabolism, cellular integrity, cell death, growth and differentiation (Hoffman, 1964; Harold, 1986; Hager et al, 1991; Yenush et al, 2002, 2005; Adams et al, 2006). Although the basic mechanisms of monovalent cation transport have been identified, our knowledge of their regulatory systems is rather fragmentary (Sanders and Bethke, 2000; Serrano and Rodriguez-Navarro, 2001; Pollard and Earnshaw, 2002). Fungi and plants share general transport mechanisms at the plasma membrane based on an H+ chemiosmotic circuit. The primary pump is the electrogenic H+-pumping P-ATPase that generates an electrochemical H+ gradient (Serrano, 1989; Sussman and Harper, 1989; Morsomme and Boutry, 2000). This H+ gradient drives the secondary transport of K+ mediated by channels and carriers (Very and Sentenac, 2002; Rodriguez-Navarro and Rubio, 2006), as well as the uptake of nutrients by H+-symporters and the efflux of Na+ by H+-antiporters (Sanders and Bethke, 2000). In plants, the regulation of H+-ATPases and K+ channels and carriers involves changes in both expression and activity of these systems. Auxin increases the levels of H+-ATPase (Hager et al, 1991; Frías et al, 1996; Rober-Kleber et al, 2003) and inward K+ channels (Philippar et al, 1999) in elongating tissues and blue light activates the H+-ATPase of guard cells by inducing its phosphorylation (Kinoshita and Shimazaki, 1999). The fungal toxin fusicoccin activates the H+-ATPase by binding to its phosphorylated C-terminus and recruiting 14-3-3 proteins (Würtele et al, 2003). Abscisic acid inhibits the activity of the inward K+ channels of Arabidopsis guard cells (KAT1 and KAT2) through a signal transduction pathway involving reactive oxygen species and Ca2+ (Kwak et al, 2003). This hormone also represses the expression of the outward K+ channel (SKOR) at the root pericycle and xylem parenchyma of Arabidopsis (Gaymard et al, 1998). K+ starvation induces the expression of high-affinity K+ carriers such as Hak5 (Maathuis et al, 2003; Shin and Schachtman, 2004; Gierth et al, 2005) by a mechanism including hydrogen peroxide production (Shin and Schachtman, 2004). Again, the signal transduction pathways of these regulations remain largely unknown. Na+ efflux in Arabidopsis is mediated by the SOS1 H+-antiporter, a transporter activated by the SOS2/CIP24-SOS3/CBL4 protein kinase–calcium sensor complex (Qiu et al, 2002; Quintero et al, 2002). Another protein kinase–calcium sensor complex, namely CIPK23-CBL1 (or CBL9), has recently been described to activate the AKT1 K+ uptake channel of Arabidopsis roots (Xu et al, 2006). These two protein kinases are the only regulatory components of plant cation homeostasis characterized at the molecular level. One approach to identify novel regulators of cation transport consists of the identification of genes that upon gain of function improve tolerance to toxic cations (Serrano et al, 1999). Gain-of-function mutants obviate genetic redundancy and can identify bottlenecks in biological pathways. We have screened the collection of 'activation-tagging' Arabidopsis mutants (Weigel et al, 2000) for tolerance to the toxic cation norspermidine (Hamana et al, 1989), and report here that the QSO2 gene (At1g15020), encoding a quiescin-sulfhydryl oxidase (QSO), is a novel regulator of monovalent cation transport at the root symplast–xylem interface. QSOs are animal and plant enzymes proposed to participate in oxidative folding of disulfide-containing secreted proteins. Quiescins have been implicated in the regulation of growth and the elaboration of extracellular matrix of animal cells (Coppock et al, 1998; Thorpe et al, 2002). Our work suggests that these eukaryotic proteins may also regulate ion homeostasis. Results A screening based on tolerance to norspermidine resulted in the par1-1D mutant We have screened the 'activation-tagging' mutant collection of the plant Arabidopsis thaliana (Weigel et al, 2000) for tolerance to toxic cations. Previous screenings for tolerance to Na+ in Arabidopsis resulted in mutants defective in either abscisic acid biosynthesis or perception. The high concentrations of NaCl required for toxicity during the germination assay (greater than 0.1 M) have significant osmotic effects, which trigger the biosynthesis of abscisic acid and this hormone inhibits germination and early growth (González-Guzmán et al, 2002). Alternatively, polyamines are toxic at millimolar concentrations, which pose no osmotic stress, and the presence of several positive charges per molecule makes their uptake very sensitive to membrane potential, negative inside. Norspermidine is a non-metabolizable polyamine (Hamana et al, 1989), and this type of toxic cation has been successfully used to demonstrate changes in membrane potential in yeast mutants affected in K+ transport (Forment et al, 2002). We have screened 16 398 lines selecting for tolerance to 3.2 mM norspermidine, a concentration that completely inhibits growth of wild-type seeds. The most resistant mutant was named par1-1D, from 'polyamine resistant', gene 1, allele 1, Dominant (Meinke and Koornneef, 1997). As indicated in Figure 1A and B, par1-1D seeds germinate and grow better than wild-type plants on plates containing norspermidine, spermidine, Li+ or Na+. Experiments in soil with adult plants also demonstrate tolerance to Li+ and Na+ (Figure 2). This tolerance is reflected both in the total size (Figure 2A) and in the discoloration of the leaves (Figure 2B and C) of stressed plants. Therefore par1-1D seems to have a pleiotropic phenotype of tolerance to toxic cations. Figure 1.Seedlings of the par1-1D mutant are more tolerant to toxic cations than wild type (wt). (A) Percentage of seeds that germinated and developed green cotyledons 6 days after sowing in MS medium supplemented with the indicated toxic cation. Error bars represent the s.d. of three independent experiments, with three replicates each (n=9). *P<0.05 and **P<0.01 by Student's test. (B) Representative seedlings from the experiment in panel A. The concentrations of toxic cations were as follows: none (control), norspermidine (2.8 mM), spermidine (7 mM), lithium (20 mM LiCl) and sodium (140 mM NaCl). Sucrose concentration was 3%, except in the case of NaCl, where 1% was utilized. Controls were sown in media with the two sucrose concentrations. Scale bar corresponds to 1 mm. Download figure Download PowerPoint Figure 2.Plants of the par1-1D mutant are more tolerant to lithium and sodium than wild type (wt). (A) Plants sown in soil and irrigated with either normal solution (control) or with irrigation solution supplemented with 50 mM NaCl or 4 mM LiCl, twice a week for 25 days. Scale bar corresponds to 5 cm. (B) Leaves 6 days after treatment with 2 mM LiCl. (C) Rosettes 11 days after treatment with 200 mM NaCl. Plants in panels B and C were sown in soil and the treatment started when the rosette was fully developed (about 20 days). Scale bar corresponds to 1 and 2 cm, respectively. Download figure Download PowerPoint The mutation was monogenic and dominant, because F1 plants exhibited tolerance to toxic cations and there was a 3:1 segregation of the phenotype in the F2 generation. Actual data were 156:44 (c2=0.96). The par1-1D mutation is caused by overexpression of At1g15020 (QSO2), a limiting factor for toxic cation tolerance Homozygous par1-1D plants were crossed to Columbia wild-type plants. From the segregating F2 generation, norspermidine-tolerant plants were selected and scored for the presence of the T-DNA by Southern blot and PCR analyses. As a result, the par1-1D mutation was shown to be linked to the T-DNA because of 60 F2 norspermidine-tolerant plants analyzed, all of them contained T-DNA (data not shown). Plasmid rescue of the T-DNA and sequencing indicated that the insertion was in the genomic region depicted in Figure 3A, with the transcriptional enhancer close to the At1g15020 gene. Northern analysis confirmed that this gene was overexpressed about three-fold in the mutant (Figure 3B, left panel). As expected, the expression of the adjacent At1g15030 gene, the promoter of which was interrupted by the insertion, was greatly decreased (Figure 3B, right panel). Figure 3.Position of T-DNA insertion in the par1-1D mutant and expression analysis of adjacent genes. (A) Location of T-DNA in the 5′ UTR region of At1g15030 gene, as determined by plasmid rescue. (B) Upper panels correspond to the Northern blot analysis of At1g15020 (left) and At1g15030 (right) mRNAs from wild type (wt) and par1-1D mutant; lower panels correspond to ethidium bromide staining of major ribosomal RNAs as loading controls. Download figure Download PowerPoint In order to test if overexpression of At1g15020 was responsible for the par1-1D phenotype, transgenic plants were generated, which overexpressed At1g15020 cDNA from the 35S promoter. Most (7 out of 11) of the transgenic lines overexpressed the Atg15020 gene (Figure 4A). Two overexpressing lines (numbers 2 and 10) were further analyzed and found to be more tolerant to toxic cations than controls (Figure 4B and C). This suggests that the par1-1D mutation was caused by overexpression of At1g15020. Figure 4.Transgenic plants overexpressing At1g15020 (QSO2) cDNA are more tolerant to toxic cations. (A) Northern blot analysis of 11 independent transgenic lines. (B) Percentage of seeds that germinated and developed green cotyledons. Lines 2 and 10 are two transgenic lines from panel A. (C) Representative seedlings from the experiment in panel B. Experiments were performed as in Figure 1, and pictures of representative seedlings were taken 6 days after sowing. Scale bar corresponds to 1 mm. Errors bars represent s.d. of three independent experiments, with three replicates each one (n=9). *P<0.05 and **P<0.01 by Student's test. Download figure Download PowerPoint At1g15020 encodes a sulfhydryl oxidase of the quiescin family and it has been previously named AtQSOX2, because there is a homologous gene, At2g01270, named AtQSOX1 (Thorpe et al, 2002). A recent review of the family (Houston et al, 2005) has reversed the numbers of the two genes. We propose the three-letter, italic name QSO2 to conform community standards for Arabidopsis genetics (Meinke and Koornneef, 1997), and maintain the numbers of the first publication (Thorpe et al, 2002). QSOs are flavoproteins containing a thioredoxin domain and which oxidize disulfhydryl groups in proteins to disulfides, with reduction of oxygen to hydrogen peroxide. They are secreted proteins located at the endoplasmic reticulum, Golgi and outside the cell. The domain structure of QSO2 is shown in Supplementary Figure 1S. QSOs are found only in multicellular organisms, plants and animals, while yeast cells contain enzymes lacking the thioredoxin domain but containing the sulfhydryl oxidase flavoprotein or ERV domain (Thorpe et al, 2002). Three T-DNA insertion mutants of At1g15020 were identified in the TAIR collection (www.arabidopsis.org) and the homozygous lines named par1-2 (SALK_066130), par1-3 (SALK_025237) and par1-4 (SALK_072829). A scheme of the T-DNA insertions is presented in Figure 5A. Quantitative RT–PCR analysis indicated that QSO2 expression levels in the mutants were 13 (par1-2), 18 (par1-3) and less than 1% (par1-4) of wild type. The three mutant lines displayed more sensitivity to toxic cations than control plants, both as seedlings grown 'in vitro' (Figure 5B; details of seedlings shown for par1-2 in Figure 5C), and as plants grown in soil (details of plants shown for par1-2 in Figure 5D). Taken together with the results of the overexpression approach, these experiments demonstrate that the QSO2 sulfhydryl oxidase is a limiting factor for tolerance to toxic cations. On the other hand, a knockout mutant of the homologous gene At2g01270 has no detectable phenotype (data not shown). Figure 5.The loss-of-function mutants of QSO2 are sensitive to toxic cations. (A) Scheme of the At1g15020 gene and localization of the T-DNA insertions in the par1-2, par1-3 and par1-4 mutants. Blocks indicate exons. Nucleotide numbering begins at the ATG translation start codon. The T-DNA left border primer (pROKLbb1) that was used to localize the T-DNA insertion is indicated by an arrow. (B) Percentage of seeds that germinated and developed green cotyledons 6 days after sowing in MS medium supplemented with the indicated toxic cation. Error bars represent the s.d. of three independent experiments, with three replicates each (n=9). *P<0.05 and **P<0.01 by Student's test. (C) Representative par1-2 seedlings from the experiment in panel A. Scale bar corresponds to 1 mm. The concentrations of toxic cations were as follows: none (control), norspermidine (2.4 mM), spermidine (6 mM), lithium (18 mM LiCl) and sodium (120 mM NaCl). Sucrose concentration was 3%, except in the case of NaCl, where 1% was utilized. Controls were sown in media with the two sucrose concentrations. (D) Soil-grown plants of the par1-2 mutant are more sensitive to lithium and sodium than wild type (wt). Representative rosettes of plants irrigated with either normal solution (control) or with irrigation solution supplemented with either 2.5 mM LiCl (left panel) or 150 mM NaCl (right panel), twice a week for 14 days. Plants were sown in soil and the treatment started when the rosette was fully developed (about 20 days). Scale bars correspond to 2.5 cm. Download figure Download PowerPoint QSO2 inversely regulates the accumulation of K+ and toxic cations One mechanism of tolerance to toxic cations is based on decreasing cation accumulation. We have measured the initial rates of uptake (Figure 6A) and the final accumulation levels (Figure 6B) of norspermidine and Na+ in Arabidopsis plants (wild type, par1-1D and par1-2) incubated in liquid culture. While the initial rate of uptake (less than 20 min) was not significantly affected by either gain- or loss-of-function of QSO2, both the uptake at longer times (1 h) and the accumulation after 2 days of both toxic cations correlated inversely with QSO2 function. We have also measured the level of K+ (Figure 7A) and the initial rate of Rb+ uptake (as indication of K+ transport; Figure 7B). Again, the initial rate of uptake was not affected by QSO2 but, opposite to the results with toxic cations, both the uptake at long times and the accumulation of K+ were directly correlated with QSO2 function. Figure 6.Homeostasis of toxic cations is altered in QSO2 mutants. (A) Initial rate of uptake of norspermidine and Na+ in wild type (wt) and mutants with gain (par1-1D) and loss (par1-2) of function of QSO2. Ten-day-old plants grown in liquid culture were transferred to fresh medium supplemented with either 2.8 mM norspermidine or 120 mM NaCl. Error bars correspond to s.d.s of the mean (n=6). (B) Steady-state accumulation of norspermidine and Na+ in wild type (wt) and mutants with gain (par1-1D) and loss (par1-2) of function of QSO2. Ten-day-old plants grown in liquid culture were transferred to fresh medium that was supplemented with either 2.8 mM norspermidine or 120 mM NaCl and further incubated for 2 days. The average of three experiments, with three replicates each is shown (n=9). Errors bars represent s.d. *P<0.05 and **P<0.01 by Student's test. DW, dry weight. Download figure Download PowerPoint Figure 7.K+ homeostasis is altered in QSO2 mutants. (A) Intracellular K+ level in wild type (wt) and mutants with gain (par1-1D) and loss (par1-2/qso2) of function of QSO2. Five-day-old seedlings were grown in liquid culture for 10 days and intracellular K+ measured as indicated in Materials and methods. The average of four experiments, with three replicates each is shown (n=12). Error bars represent s.d.s. *P<0.05 by Student's test. (B) K+ transport activity was measured by the initial rate of Rb+ uptake. Ten-day-old plants grown in liquid culture were transferred to K+-free medium supplemented with 1 mM RbCl and samples taken at the indicated times for intracellular Rb+ measurement as indicated in Materials and methods. Errors bars represent s.d.s (n=6). DW, dry weight. Download figure Download PowerPoint QSO2 modulates the loading of cations into the xylem One determinant of cation accumulation, which could be affected by QSO2 is the plasma membrane electrical potential (Mulet et al, 1999). However, we have measured this parameter in root epidermal cells of wild type and plants with gain and loss of function of QSO2, and found no significant differences (Supplementary Table 1S). Another determinant of cation accumulation is the plasma membrane H+-ATPase, the primary pump that energizes all secondary transporters. However, neither the amount nor the activity of the enzyme was affected by QSO2 mutations (Supplementary Figure 2S). Also, the protein level of the root K+ uptake channel AKT1 (Hirsch et al, 1998) was not affected by QSO2 mutations (Supplementary Figure 3S A) and overexpression of QSO2 still confers tolerance to toxic cations in the akt1-1 mutant (Supplementary Figure 3S B). As QSO2 mutations affect cation accumulation at long times but not the initial rate of uptake, we hypothesized that this QSO regulates cation loading at the root xylem and subsequent cation accumulation at the shoot. The results of Figure 8A and B indicate that, effectively, QSO2 positively regulates shoot accumulation of K+, while inhibiting shoot accumulation of toxic cations. Figure 8C and D show the same effect of QSO2 in the case of xylem sap concentrations. Therefore, a plausible mechanism of QSO2 action is the activation of K+ efflux at the root symplast–xylem interface while inhibiting the efflux of toxic cations at this location. Figure 8.QSO2 positively regulates shoot and xylem accumulation of K+ while inhibiting accumulation of toxic cations. (A, B) Shoot cation content in wild type (wt) and mutants with gain (par1-1D) and loss (par1-2) of function of QSO2. Na+/K+ and Li+/K+ contents in the shoot were measured under irrigation with 50 mM NaCl or 15 mM LiCl during 5 or 3 days, respectively. Control plants were irrigated with normal solutions (control). (C, D) Cation content in xylem sap in wild type (wt) and mutants with gain (par1-1D) and loss (par1-2/qso2) of function of QSO2. Na+/K+ and Li+/K+ content in the xylem were measured under irrigation with 50 mM NaCl or 10 mM LiCl, during 3 or 2 days, respectively. The average of two experiments, with three replicates each is shown (n=6). Errors bars represent ±s.d. *P<0.05 **P<0.01 by Student's test. Download figure Download PowerPoint The skor-1 mutant is sensitive to toxic cations but overexpression of QSO2 still confers tolerance in this mutant Although QSO2 could act on several transporters, the possibility exist that its primary target is K+ efflux at the symplast–xylem interface, and that activation of this system indirectly inhibits the efflux of toxic cations. The rationale is that activation of K+ efflux would hyperpolarize the potential difference across the symplast/xylem boundary (xylem positive), and this elevated potential would inhibit the efflux of toxic cations mediated by nonspecific channels. One way to test our hypothesis was to compare the phenotypes of the par1-2- and skor-1-null mutants. SKOR is an outward K+ channel mediating K+ release into the xylem sap (Gaymard et al, 1998). As predicted by our model, the skor-1 mutant is sensitive to toxic cations in addition to K+ depletion (Figure 9A and B). Figure 9.The skor-1 mutant is sensitive to toxic cations and overexpression of QSO2 still confers tolerance in this mutant. (A) Percentage of seeds that germinated and developed green cotyledons, 5 days after sowing in MS medium supplemented with the indicated toxic cation Errors bars represent s.d. of three independent experiments, with three replicates each (n=9). Columns marked with different letters represent significantly different means according to statistical analysis (P<0.05, ANOVA, Tukey's test). (B) Representative seedlings from the experiment in panel A. Scale bar corresponds to 1 mm. The concentrations of toxic cations were as follows: none (control), norspermidine (2.4 mM), spermidine (6 mM), lithium (18 mM LiCl), sodium (120 mM NaCl) and a K+-free medium supplemented with 10 μM KCl. Sucrose concentration was 3%, except in the case of NaCl and K+-free medium, where 1% was utilized. Controls were sown in media with the two sucrose concentrations. Wt, wild-type Columbia control; 1.12 and 1.15, transgenic lines derived from the skor1-1 mutant overexpressing QSO2. Download figure Download PowerPoint Although this result can be considered as a 'proof of concept' for the mechanism of QSO2 suggested above, SKOR is not the only system loading K+ into xylem. Actually, overexpression of QSO2 still confers tolerance to toxic cations and to K+ depletion in the skor-1 mutant (Figure 9A and B). Therefore, other unknown systems involved in xylem loading of K+ must be affected by QSO2. Characterization of QSO2 expression and encoded protein Quantitative RT–PCR analysis of Arabidopsis tissues indicates that QSO2 is mostly expressed in roots (Figure 10A). Transcription in roots is induced about two-fold by K+ depletion (Figure 10B), and this regulation is in agreement with the proposed activation by QSO2 of systems loading K+ into the xylem. Figure 10.Expression analysis of QSO2. (A) Quantitative RT–PCR analysis of the QSO2 gene expression pattern in different organs. Values are mean ΔCt±s.d. (right) and relative transcript levels (left) were calculated as 2−ΔCt. (B) Induction of QSO2 by K+ starvation. Seedlings of wild-type Arabidopsis (Columbia) were treated with either 2.8 mM norspermidine, 25 mM LiCl, 120 mM NaCl, 10 mM H2O2, 10 μM ABA or K+-free medium for 16 h, as indicated. Quantitative RT–PCR analyses were conducted in three independent biological experiments. Expression values are relative to those of non-stressed control seedlings (taken as 1). Download figure Download PowerPoint The localization of expression of more than 22 000 genes in the Arabidopsis root has been described by Birnbaum et al (2003). QSO2 (At1g15020) behaves as a housekeeping gene, because it is expressed at similar levels (within a factor of 2) in different root tissues (stele, endodermis, cortex and epidermis) and zones (meristematic, elongation and mature zone with root hairs). A QSO2∷GFP fusion transiently expressed in Nicothiana benthamiana epidermal cells is present at the cell surface (Figure 11A), as expected from its N-terminal signal peptide. Plasmolysis of cells indicates that the fusion protein is associated with the cell wall and not the plasma membrane (Figure 11B). After expression in yeast about 30% of the recombinant QSO2 protein is solubilized by digestion of the cell wall in the process of making yeast protoplasts (Supplementary Figure 4S B). This fraction corresponds to the extracellularly secreted form of the enzyme. Figure 11.A QSO2∷GFP fusion protein is secreted at the cell wall. (A) Laser scanning confocal microscopy image (LSCM) of N. benthamiana epidermal cells expressing a QSO2:GFP fusion, showing GFP fluorescence along the cell periphery. (B) LSCM of plasmolyzed epidermal cells expressing QSO2:GFP fusion, showing GFP fluorescence in the cell wall. Left, fluorescent image; right, bright-field image. Arrows indicate plasma membrane separated from cell wall. Scale bar corresponds to 20 μm. Download figure Download PowerPoint We have purified QSO2 after expression in yeast (Supplementary Figure 4S B). Assay of sulfhydryl oxidase activity demonstrated specificity for dithiol compounds such as dithioerithrytol and dithiothreitol, and insignificant activity (less than 1%) with monothiols such as mercaptoethanol and glutathione. The turnover number measured with simple dithiols (14 min−1) is within the range of values detected for sulfhydryl oxidases (Levitan et al, 2004). The optimum pH for activity is 7.5, but it has considerable activity (40–60% of optimum) at the pH values reported to prevail at the apoplast (from 5.5 to 6.5; Gao et al, 2004). Discussion QSOs are animal and pl
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