AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana Abstract The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs. Introduction Glutathione is a sulfur-containing tripeptide comprised of l-γ-glutamyl-l-cysteinyl-glycine, which performs various important roles in plant cells such as redox homeostasis, detoxification of heavy metals and storage of organic sulfur (Noctor et al. 2011). Glutathione within the cell is primarily maintained in the reduced state (GSH), which is converted to the oxidized state (GSSG) in buffering reactive oxygen species, but in most cases is converted back to GSH by glutathione reductase. GSH is also involved in regulation of protein activity by changing protein thiol status (Foyer and Noctor 2005). Phytochelatins (PCs), the polymerized form of GSH, can detoxify heavy metals and metalloids such as cadmium (Cd) and arsenic (As) by chelating them (Cobbett 2000, Mendoza-Cózatl et al. 2011). GSH is also involved in regulation of plant growth, such as flowering (Ogawa et al. 2001, Yanagida et al. 2004, Hatano-Iwasaki and Ogawa 2007) and cell division (Vernoux et al. 2000, Vivancos et al. 2010a, Vivancos et al. 2010b). GSH functions as a major storage transport form of organic sulfur in plants. Sulfur is taken up from the environment into root cells mainly as sulfate, and transported into chloroplasts for reduction to sulfite then sulfide. Sulfide is assimilated into cysteine by reacting with O-acetyl-l-serine. However, plants primarily use GSH as a form of organic sulfur (Leustek et al. 2000). GSH is synthesized from the constituent amino acids in a two-step reaction by γ-glutamyl-cysteine synthetase (Hell and Bergmann 1990) and GSH synthetase (Wang and Oliver 1996), which are encoded by single genes GSH1 (May and Leaver 1994) and GSH2 (Wang and Oliver 1996), respectively. The evidence that GSH is the major form of organic sulfur stored and transported from sink to source organs was provided in experiments using Nicotiana tabacum fed with 35S-labeled sulfate (Rennenberg et al. 1979). Kuzuhara et al. (2000) observed that the GSH concentration in phloem sap taken from Oryza sativa using the insect laser method was around 5 mM, which is several times higher than the sulfate concentration, suggesting that GSH is the main form of sulfur transported through the phloem. GSH translocated through the phloem was also shown to be a signal to regulate sulfate uptake and ATP sulfurylase activity in Brassica napus (Lappartient and Touraine 1996). Although long-distance transport of GSH through the phloem is important for distribution of sulfur in plants, little is understood about the transporters involved in this process. AtMRP1 and AtMRP2 were identified as transporters for GSH and GSH conjugates into plant vacuoles, and AtCLTs were shown to transport GSH from chloroplasts into the cytosol (Lu et al. 1997, Lu et al. 1998, Maughan et al. 2010, Bachhawat et al. 2013). However, none of these transporters was implicated in the transport of GSH through the phloem. In Saccharomyces cerevisiae, Hgt1 was identified as a high-affinity GSH transporter (Km = 54 µM), and a yeast hgt1Δ mutant was unable to grow on a medium with GSH as a sulfur source (Bourbouloux et al. 2000). Hgt1 belongs to the oligopeptide transporter (OPT) family with 12–14 transmembrane domains. The Arabidopsis genome encodes nine possible OPT homologs, named AtOPT1–AtOPT9 (Koh et al. 2002). Expression of AtOPT1, 4, 5, 6 and 7 in yeast conferred the ability to take up tetra- and pentapeptides (Koh et al. 2002). Cagnac et al. (2004) observed that complementation of the yeast hgt1Δ mutant by AtOPT6 restored growth on a medium with GSH or GSSG as the sole sulfur source, and induced [3H]GSH transport into cells. Reporter gene experiments showed that AtOPT6 is mainly expressed in the vascular bundles of leaves, petioles and stems in plants (Cagnac et al. 2004, Stacey et al. 2006), suggesting involvement of AtOPT6 in GSH transport through the vascular tissue. In unfertilized gynoecium in flowers, expression of AtOPT6 was high in ovules, indicating a possible role for AtOPT6 in ovule development (Stacey et al. 2006). Pike et al. (2009) demonstrated that AtOPT6 expressed in Xenopus laevis oocytes transported GSH and PC2, but the Km for GSH was 566 µM, which was much higher than those for other peptides such as 10.8 µM for KLLLG and 71 µM for AtCLE19p12. Hence, the authors suggested that AtOPT6 may mediate the movement of signaling peptides. Recently, Zhang et al. (2016) reported that AtOPT4 complemented a yeast hgt1Δmet15Δ mutant for growth on a medium with GSH as the sole sulfur source and induced [35S]GSH transport into cells. The double knockout mutant opt2/opt4 in Arabidopsis showed a reduced content of GSH in siliques, suggesting a possible role for AtOPT4 in transporting GSH to siliques (Zhang et al. 2016). AtOPT3 was proposed to mediate shoot to root signaling of the Fe/Zn/Mn status for maintenance of these trace elements and it was also reported that misregulation of AtOPT3 led to an overaccumulation of cadmium in seeds (Stacey et al. 2008, Mendoza-Cózatl et al. 2014). A few OPT transporters have been studied in plants other than Arabidopsis. For example, BjGT1 from Brassica juncea (Bogs et al. 2003) and OsGT1 from Oryza sativa (Zhang et al. 2004) were also shown to complement a hgt1Δ mutant for growth with GSH as the sole sulfur source and induce [3H]GSH transport into cells. In the present study, we investigated the involvement of AtOPT6 in long-distance GSH transport in planta by analyzing the phenotype of Atopt6 mutant plants. Results Glutathione content in source and sink organs in the T-DNA insertional mutants of OPT6 In order to study the functions of AtOPT6 in planta, we obtained the T-DNA insertional mutant GK-029H12 which results from T-DNA insertion in the second exon of the AtOPT6 gene (At4g27730) (Supplementary Fig. S1A). We confirmed the lack of measureable OPT6 transcript in the homozygous GK-029H12 line by reverse transcription–PCR (RT–PCR; Supplementary Fig. S1B). To compare the distribution of GSH in whole plants, GK-029H12 and the corresponding wild-type (Columbia-0) plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) in normal MGRL hydroponic solution (Fujiwara et al. 1992, Hirai et al. 1995) followed by determination of the GSH content in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm in length) (Fig. 1A). There was no visible phenotype in the mutants compared with wild-type plants after 8 weeks of growth post-germination. The GSH content in flowers and siliques was decreased significantly in GK-029H12 mutant plants compared with wild-type plants, while there was no difference in GSH content in leaves and stems (Fig. 1A). This suggests that the GSH synthesis capacity in source leaves was not inhibited but translocation of GSH from source to sink organs was reduced in the mutant plants. Fig. 1 View largeDownload slide GSH concentration in the wild type (WT), and GK-029H12 and GK-448H05 mutants, and accumulation of AtOPT6 mRNA in WT plants. Plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark). GSH contents in roots, rosette leaves, cauline leaves, stems, flowers and siliques in GK-029H12 (A) or GK-448H05 (B) together with those in WT plants were determined. Means ± SDs of four biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). In (C), the levels of AtOPT6 mRNA in roots, rosette leaves, cauline leaves, stems, flowers and green siliques were determined in WT plants and normalized to those of ACTIN8. Means ± SDs of three biological replicates are shown, with the mean in roots set as 1. Fig. 1 View largeDownload slide GSH concentration in the wild type (WT), and GK-029H12 and GK-448H05 mutants, and accumulation of AtOPT6 mRNA in WT plants. Plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark). GSH contents in roots, rosette leaves, cauline leaves, stems, flowers and siliques in GK-029H12 (A) or GK-448H05 (B) together with those in WT plants were determined. Means ± SDs of four biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). In (C), the levels of AtOPT6 mRNA in roots, rosette leaves, cauline leaves, stems, flowers and green siliques were determined in WT plants and normalized to those of ACTIN8. Means ± SDs of three biological replicates are shown, with the mean in roots set as 1. In order to confirm that the decreased GSH levels in sink organs were caused by the lack of OPT6 transcript, another T-DNA insertional mutant line, GK-448H05, was included in this analysis. The GK-448H05 mutant results from T-DNA insertion in the second exon of the AtOPT6 gene, and we confirmed the lack of measureable AtOPT6 transcript in the homozygous GK-448H05 line by RT–PCR (Supplementary Fig. S1C). As shown in Fig. 1B, GSH concentrations in sink organs such as siliques and flowers were significantly decreased in GK-448H05 compared with those in wild-type plants. Although absolute values of concentrations were different in Fig. 1A and B, which is probably due to difference in environmental conditions since the plants used for Fig. 1A and B were cultured independently, values of GSH of mutants relative to wild-type plants in flowers and siliques were comparable, 87% and 71% in GK-029H12 (Fig. 1A), and 61% and 73% in GK-448H05, respectively. The decrease in GSH levels in sink organs in the opt6 mutants indicates that OPT6 contributes the most in these organs. To confirm this, OPT6 gene expression was analyzed in various organs in wild-type plants. Total RNA was extracted from roots, rosette leaves, cauline leaves, stems, flowers and siliques of wild-type plants hydroponically grown for 8 weeks as above and OPT6 mRNA accumulation was determined by real-time PCR. As shown in Fig. 1C, expression of OPT6 in flowers and siliques was highest among the tissues examined, >3-fold higher than that in rosette leaves and >2-fold higher than that of cauline leaves. These expression patterns of OPT6 suggest that OPT6 protein contributes the most in sink organs, which is nicely correlated with the significant decrease in GSH concentration in flowers and siliques of the mutant plants (see Fig. 1A, B). Glutathione content in phloem sap of the opt6 mutant plants Previously published reports demonstrated that expression of AtOPT6 is mainly in vascular tissues (Cagnac et al. 2004, Stacey et al. 2006). Given these observations and the reduction of GSH translocation from source to sink organs in opt6 mutants (Fig. 1), we hypothesized that loading GSH into phloem was decreased by the mutations in the AtOPT6 gene. To confirm this, GSH contents in phloem sap in the GK-029H12 mutant were compared with those in wild-type plants. Plants were hydroponically grown for 8 weeks in the same way as used for GSH analysis above. To extract phloem sap, petioles were cut from rosette leaves, and were then soaked in EDTA solution for 1 h followed by soaking in distilled water for 8 h under darkness as described in Tetyuk et al. (2013). In order to normalize for the efficiency of extraction of phloem sap, GSH concentrations in the phloem sap were normalized to the glutamate concentration determined in the same samples. Previously, Cagnac et al. (2004) reported that GSH transport by AtOPT6 is inhibited by glutamine but not by glutamate, suggesting that AtOPT6 may transport glutamine but not glutamate, thus the concentration of glutamate was used for normalization. GSH concentrations before normalization and concentrations of major amino acids are presented in Supplementary Table S1. As noted, glutamate concentrations were not significantly different between mutants and the wild type. As shown in Fig. 2, the GSH contents in phloem sap normalized to glutamate content were not significantly different between GK-029H12 and wild-type plants. As a decrease of phloem GSH was not observed in the mutant, it is considered that OPT6 is not involved in loading of GSH into the phloem in source organs. Fig. 2 View largeDownload slide GSH concentrations normalized to glutamate concentrations in phloem sap from the wild type (WT) and GK-029H12 mutants. Phloem sap was extracted from rosette leaves of plants hydroponically grown for 8 weeks. GSH concentrations are normalized to glutamate concentrations. Values are (GSH concentrations/glutamate concentrations)×100. The means ± SDs of four biological replicates are shown. Fig. 2 View largeDownload slide GSH concentrations normalized to glutamate concentrations in phloem sap from the wild type (WT) and GK-029H12 mutants. Phloem sap was extracted from rosette leaves of plants hydroponically grown for 8 weeks. GSH concentrations are normalized to glutamate concentrations. Values are (GSH concentrations/glutamate concentrations)×100. The means ± SDs of four biological replicates are shown. The opt6 mutant plants show a delay in flowering Since GSH was reported to be involved in the transition from the vegetative rosette stage to the reproductive growth stage (Ogawa et al. 2001, Yanagida et al. 2004, Hatano-Iwasaki and Ogawa 2007), we examined the time of flowering in opt6 mutants relative to the wild type. GK-448H05, GK-029H12 and wild-type plants were grown hydroponically on rockwool under long-day conditions (16 h light/8 h dark). As shown in Fig. 3, bolting was delayed in both mutant lines compared with wild-type plants. The time at which >50% of total plants (n = 20) bolted was 30, 33 and 36 d after germination for the wild type, GK-448H05 and GK-029H12, respectively. Fig. 3 View largeDownload slide Timing of bolting in the wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were grown hydroponically on rockwool. The cumulative number of plants bolted from 27 to 39 d after germination was counted each day; n = 20. Fig. 3 View largeDownload slide Timing of bolting in the wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were grown hydroponically on rockwool. The cumulative number of plants bolted from 27 to 39 d after germination was counted each day; n = 20. The number of rosette leaves on bolting plants, which is an indicator of timing of bolting (Ogawa et al. 2001), was also counted. As shown in Table 1, both GK-448H05 and GK-029H12 mutant plants had an increased number of rosette leaves compared with wild-type plants, consistent with the delay in flowering shown by the mutant plants. A representative picture of the wild-type and opt6 mutants undergoing bolting is shown in Supplementary Fig. S2. Table 1 The number of rosette leaves on bolting plants   Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**    Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**  Values are means ± SD (n = 20). Asterisks indicate a significant difference at *P < 0.05 and *P < 0.01 (Student’s t-test), compared with the wild type. Table 1 The number of rosette leaves on bolting plants   Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**    Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**  Values are means ± SD (n = 20). Asterisks indicate a significant difference at *P < 0.05 and *P < 0.01 (Student’s t-test), compared with the wild type. The opt6 mutant seeds show lower germination rates Since expression of AtOPT6 is high in ovules (Stacey et al. 2006), AtOPT6 was suggested to be involved in seed development. To examine this, seeds in young and mature siliques were observed under a stereo microscope. Seeds of GK-448H05 and GK-029H12 mutant plants were normal and there was no apparent difference compared with wild-type seeds both in young green siliques and in mature dried siliques (Supplementary Fig. S3). To confirm proper maturity of seeds, the germination rate was compared between the opt6 mutants and wild-type plants. Twenty seeds per agar plate were sown in triplicate, and germination rates were calculated for each plate. As shown in Table 2, the germination rates of GK-448H05 and GK-029H12 seeds were less than half compared with that of wild-type plants. This suggests that lack of OPT6 inhibited proper seed maturation. Although germination rates were lower in the mutants, the rate of growth of the germinated plants was roughly equivalent to that of the wild type (data not shown). Table 2 The germination rates on agar plates   Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*    Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*  Values are means ± SD (n = 3). Asterisks indictates a significant difference at P < 0.01 (Student’s t-test) compared with the wild type. Table 2 The germination rates on agar plates   Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*    Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*  Values are means ± SD (n = 3). Asterisks indictates a significant difference at P < 0.01 (Student’s t-test) compared with the wild type. Phytochelatin and Cd distribution in the opt6 mutants after Cd treatment Cd is thought to be transported through phloem complexed with GSH or PCs (Mendoza-Cózatl et al. 2011). Therefore, we also investigated involvement of OPT6 in Cd distribution using GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown under long-day conditions for 8 weeks, during which they were exposed to Cd for 4 d. GSH, PC2, PC3 and PC4 contents in source and sink organs were determined. No visible symptom of Cd stress was observed in any genotype at sampling time. As shown in Fig. 4B–D, PC2 content was higher than that of PC3 or PC4 in most organs, which suggests that PC2 is the major PC accumulated by Cd treatment in our conditions. The GSH concentration in rosette leaves was higher in opt6 mutants than that in wild-type plants (Fig. 4A). Concentrations of PC2 in flowers were significantly lower in both mutant lines compared with those in wild-type plants (Fig. 4B), suggesting that lack of AtOPT6 leads to a decrease in PC2. In source organs, such as rosette leaves, no differences in PC2 content were observed between the opt6 mutants and wild-type plants. PC3 and PC4 contents were not changed in rosette leaves, but in sink organs, such as siliques, PC4 levels were decreased in both mutant lines compared with wild-type plants. Fig. 4 View largeDownload slide GSH and PC concentrations in wild-type (WT), GK-448H05 and GK-029H12 mutant plants after Cd treatment. Plants hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) were treated with 10 μM Cd for 4 d. GSH (A), PC2 (B), PC3 (C) and PC4 (D) contents in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm) were determined. Means ± SDs of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05, *P < 0.1). Fig. 4 View largeDownload slide GSH and PC concentrations in wild-type (WT), GK-448H05 and GK-029H12 mutant plants after Cd treatment. Plants hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) were treated with 10 μM Cd for 4 d. GSH (A), PC2 (B), PC3 (C) and PC4 (D) contents in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm) were determined. Means ± SDs of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05, *P < 0.1). If PCs are involved in Cd movement, then the distribution of Cd should reflect changes in PC content. Plants hydroponically grown and treated with Cd at the same time as those used for PC determination were subjected to Cd analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES). To obtain sufficient tissue for the measurement, flowers and siliques were pooled as sink tissues and used for a single Cd determination. As shown in Fig. 5, Cd content was highest in roots relative to other organs. In roots, Cd concentrations were >2-fold higher both in GK-448H05 and GK-029H12 compared with wild-type plants (Fig. 5A). These results suggest that lack of functional AtOPT6 caused accumulation of Cd in roots. Fig. 5 View largeDownload slide Cd concentration in wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown and treated with Cd at the same time as those used for Fig. 5. Cd concentrations in roots (A) cauline leaves, stems, flowers and green siliques (1–2 cm) (B) were determined. Means ± SD of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). Fig. 5 View largeDownload slide Cd concentration in wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown and treated with Cd at the same time as those used for Fig. 5. Cd concentrations in roots (A) cauline leaves, stems, flowers and green siliques (1–2 cm) (B) were determined. Means ± SD of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). Discussion GSH levels in sink organs such as flowers and siliques were decreased in opt6 mutants compared with wild-type plants (Fig. 1A, B). Absolute values of GSH content varied between Fig. 1A and B, which we attribute to different growth conditions since the plants used for Fig. 1A and B were grown independently. GSH concentrations are known to fluctuate depending on slight changes in environmental conditions. For example, GSH contents in shoots of Arabidopsis wild-type (Col-0) plants grown on normal MGRL agar plates for 10 d were about 500 nmol g FW−1 in Maruyama-Nakashita et al. (2006) and 250 nmol g FW−1 in Yamaguchi et al. (2016). In our analysis, the values of mutants and wild-type plants grown in the same culture were compared and the values of GSH of mutants relative to wild-type plants in flowers and siliques were similar in GK-029H12 and GK-448H05. This suggests that AtOPT6 is involved in long-distance transport of GSH from source to sink organs. As GSH is the major transport form of sulfur (Rennenberg et al. 1979), AtOPT6 may have an important role in distribution of sulfur in plants. Transport of GSH to sink organs was not completely inhibited in opt6 mutants, implying that there may be other routes for GSH transport. AtOPT2 and AtOPT4 are likely candidates since opt2/opt4 double mutant plants showed reduced GSH concentration in siliques compared with wild-type plants (Zhang et al. 2016). AtOPT6 is mainly expressed in plant vascular tissues (Cagnac et al. 2004, Stacey et al. 2006). Hence, we hypothesized that transport of GSH through the phloem from source to sink tissues was decreased in opt6 mutants. However, the GSH content in phloem sap was not changed in opt6 mutant plants. This suggests that loading of GSH into the phloem is not inhibited in opt6 mutant plants (Fig. 2). Cagnac et al. (2004) observed in reporter gene experiments that expression of AtOPT6 was high in the cambial zone of the vascular bundles in the petiole and stem of adult plants. The cambial zone between the phloem and xylem consists of meristematic cells where nutrients are required for actively dividing cells. After unloading from phloem companion cells in sink organs, solutes are transported into the apoplast and then incorporated into cells surrounding the phloem. We propose that AtOPT6 is the transporter for incorporating GSH into cells surrounding the phloem such as cells in the cambial zone from the apoplastic space. In our experiments, OPT6 mRNA expression was higher in sink organs such as flowers and siliques compared with leaves, stems and roots (Fig. 1C), which is in agreement with the findings of Koh et al. (2002) and also supports the idea that AtOPT6 functions in absorbing GSH into cells in sink organs transported through the phloem from source organs. The time of bolting for opt6 mutant plants was delayed (Fig. 3; Table 1), suggesting that AtOPT6 contributes to the transition from vegetative to reproductive growth. Two possible mechanisms may explain the contribution of AtOPT6 to flowering; one is through transportation of GSH and another is through transportation of other peptides. The Arabidopsis cad-1 mutant has reduced amounts of GSH due to a mutation in the γ-glutamyl-cysteine synthetase gene and also displays delayed flowering. This phenotype is stronger when an inhibitor of γ-glutamyl-cysteine synthetase is added and is abolished by application of GSH (Ogawa et al. 2001). In Eustoma grandiflorum, induction of bolting by vernalization was nullified by inhibition of GSH synthesis, while addition of GSH induced bolting (Yanagida et al. 2004). Hatano-Iwasaki and Ogawa (2007) suggested that a crucial amount of GSH synthesized during early growth is important for promotion of flowering. Our results, together with those of previous studies, suggest the possibility that GSH incorporated into sink organs by AtOPT6 is involved in regulation of flowering time. Alternatively, we cannot exclude the possibility that transport of other, unidentified peptides by AtOPT6 contributes to flowering time regulation. Pike et al. (2009) reported that AtOPT6 expressed in X. laevis oocytes transported not only GSH but also other peptides, including some CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptides that are known to be mobile signals controlling cell differentiation and plant development. It is possible that the delay in bolting is influencing the amount of GSH and PC in flowers and siliques. However, we view this as less likely because a special effort was made to sample flowers and siliques at identical developmental stages, opened flowers and green siliques 1–2 cm in length, respectively. The seed germination rate was reduced more than half in both mutant lines compared with the wild type (Table 2), indicating that seed development was inhibited by the lack of AtOPT6. Although the appearance of seeds from the wild-type and mutant plants was not different (Supplementary Fig. S3), it is likely that their metabolism differs. Cairns et al. (2006) showed that a functional GSH1 was required for maturation of Arabidopsis seeds. Our results coupled with the report of Cairns et al. (2006) suggest that GSH is a key metabolite contributing to seed maturation. Stacey et al. (2006) observed that expression of AtOPT6 was high in ovules in unfertilized gynoecium in flowers, thus it is possible that AtOPT6 contributes not only to seed maturation but also to development of ovules. Recently Giaretta et al. (2017) demonstrated the roles of the γ-glutamyl transferases GGT1 and GGT2 in delivery of cysteine in seeds. GGT1 and GGT2 are both located in the apoplast and involved in GSH degradation (Ohkama-Ohtsu et al. 2007a). Giaretta et al. (2017) suggested that GSH transported from leaves to seeds through the phloem followed by unloading is released to the apoplast of the outer and inner integument, where GGT1 is strongly expressed and may assist in GSH degradation to provide cysteine to the endosperm. GGT2 expression is high in the suspensor and micropylar endosperm during embryo development and may function in supplying cysteine to the embryo from the micropylar endosperm. In their study, silencing of both ggt1 and ggt2 by RNA interference resulted in low seed yields. They concluded that the extracellular GGTs integrate sulfur levels when demand is high by degrading GSH. It is possible that OPT6 and GGTs co-ordinately function in delivery of sulfur into seeds, where OPT6 takes up GSH unloaded from the phloem into cells of ovules followed by releasing it into apoplast of the outer and inner integument, and degradation into cysteine by GGT1 and GGT2. In opt6 mutant plants, PC content after Cd treatments was also lower in flowers compared with wild-type plants (Fig. 4). PC2 was the major PC accumulated, relative to PC3 or PC4. The concentration of PC2 in flowers was significantly lower in both opt6 mutants compared with wild-type plants (Fig. 4B). Although not as significant, the content of PC3 and PC4 also tended to be lower in opt6 mutants (Fig. 4C, D). Pike et al. (2009) reported that AtOPT6 expressed in X. laevis oocytes allowed transport of PC2. Thus, we propose that AtOPT6 transports PCs in addition to GSH, probably into cells surrounding the phloem in sink organs. Cd accumulated in roots in both GK-448H05 and GK-029H12 mutant plants, relative to wild-type plants (Fig. 5A). Cd is thought to be transported in the phloem complexed with GSH or PCs (Mendoza-Cózatl et al. 2011). Cagnac et al. (2004) showed that yeast hgt1Δ mutant cells complemented with AtOPT6 had the ability to transport GS conjugates. They also observed that complementation with AtOPT6 increased yeast sensitivity to Cd. This sensitivity was enhanced when GSH was added to the medium. The authors suggested that AtOPT6 transports Cd conjugated with GSH (Cagnac et al. 2004). As expression of OPT6 is high in flowers compared with other organs (Fig. 1C), it is possible that AtOPT6 functions in unloading of GSH–Cd conjugates and PC–Cd conjugates from the phloem into flowers and siliques. Mendoza-Cózatl et al. (2011) suggested that GSH–Cd conjugates and PC–Cd conjugates loaded into the phloem are more likely to be sequestered in root vacuoles by the PC transporters ABCC1 and ABCC2, which is supported by measurements of the transcript levels of ABCC1 and ABCC2, which are on average 3-fold higher in roots compared with those in shoots (Mustroph et al. 2009). It is possible that inhibition of unloading into flowers and siliques in the opt6 mutants further enhanced the movement of thiol–Cd complexes in phloem towards roots for sequestering into the root vacuoles, which caused Cd accumulation in roots in the mutants (Fig. 5A). Although unloading of thiol–Cd complexes appears to be inhibited in the mutants, the concentration of Cd in flowers and siliques was not changed in GK-448H05 and was only slightly higher in GK-029H12 compared with wild-type plants. This suggests that another route probably exists for Cd entering into flowers independently of AtOPT6. However, in general, flowers and siliques are not major sinks for Cd given the very low concentrations measured in these organs. Recently another gene in the OPT family in Arabidopsis, AtOPT3, was shown to be involved in Cd distribution in plants (Mendoza-Cózatl et al. 2014). The opt3 mutant plants accumulated Cd in roots and seeds, and underaccumulated Cd in leaves, while they accumulated Fe and Zn in both roots and leaves compared with wild-type plants. AtOPT3 was suggested to function in signaling of Fe status from shoot to roots. However, the role of AtOPT3 in Cd movement may be independent of Fe signaling because the pattern of Fe distribution in the opt3 mutant was different from that of Cd (Stacey et al. 2008, Mendoza-Cózatl et al. 2014). These previous studies with AtOPT3 and our results with AtOPT6 demonstrated that transporters in the OPT family play key roles in Cd movement in plants. In conclusion, our present study together with those of Koh et al. (2002), Cagnac et al. (2004), Stacey et al. (2006) and Pike et al. (2009) suggest that AtOPT6 transports GSH, and PCs and Cd complexed with these thiols into actively dividing cells around phloem in sink organs. In this way, AtOPT6 contributes to the distribution of organic sulfur and GSH incorporated into sink organs, which in turn is critical for maintenance of redox homeostasis of cells, as well as for the myriad of reactions that require organic sulfur. However, as indicated by Cagnac et al. (2004) and Pike et al. (2009), AtOPT6 appears to have broad specificity with regard to peptide transport and, hence, may play additional roles beyond the movement of GSH and PCs in planta. Materials and Methods Plant materials The GK-029H12 mutant (ecotype Columbia-0) was provided by GABI-kat (http://www.gabi-kat.de/) (Rosso et al. 2003). The GK-029H12 line is a result of a T-DNA insertion in the second exon of the AtOPT6 gene. The homozygous plants were screened by PCR using the gene-specific primers GK-029-F (5'-CTCATGCCTCCTCTTGATTTC-3') and GK-029-R (5'-CTTCAACGTCAAGGAACATG-3'), and the T-DNA left-border primer GABI-8474 (5'-ATAATAACGCTGCGGACATCTACATTTT-3') (http://www.gabi-kat.de/). A lack of amplifiable transcript of AtOPT6 in the GK-029H12 mutant was confirmed by quantitative RT–PCR using primers OPT6RT-F (5'-GAAGAGCTGACTAGAGAAGATAGG-3') and OPT6RT-R (5'-ACCACTCAGATTCTTGGATTTG-3'). The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). The GK-448H05 mutant (ecotype Columbia-0) was provided by GABI-kat (http://www.gabi-kat.de/) (Rosso et al. 2003). The GK-448H05 line is a result of a T-DNA insertion in the second exon of the AtOPT6 gene. The homozygous plants were screened by PCR using the gene-specific primers GK-448-F (5'-AACATGATAAGCGCGATA-3') and GK-448-R (5'-AGTCTCTCTCTTTGGTGCGTTACA-3'), and the T-DNA left-border primer GABI-8474 (http://www.gabi-kat.de/). A lack of amplifiable transcript of AtOPT6 in the GK-448H05 mutant was confirmed by quantitative RT–PCR using primers OPT6RT-F and OPT6RT-R described above. The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). RT–PCR Total RNA was treated with DNase I (Thermo Fisher Scientific, https://www.thermofisher.com/us/en/home.html) and used as a template to synthesize cDNA with SuperScrpt III reverse transcriptase (Thermo Fisher Scientific) and oligo(dT)20 according to the manufacturer’s instructions. PCR analysis was performed with KOD plus NEO (TOYOBO Life Science, http://lifescience.toyobo.co.jp). Quantitative real-time PCR analysis Real-time PCR analysis was performed with LightCycler® Nano (Roche, https://lifescience.roche.com/shop/home) and Essential DNA Green Master (Roche) as recommended by the manufacturer. The primers used for quantifying the OPT6 transcripts were 5'-TCAACGGAAGTGACTTGTGG-3' and 5'-ACCACCACAATGGAACTTCC-3'. The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). Hydroponic culture of plants Unless otherwise indicated, Arabidopsis thaliana plants were grown hydroponically for 8 weeks in 50 ml conical tubes at 22°C under 16 h light (150 μmol m−2 s−1)/8 h dark as described in Ohkama-Ohtsu et al (2007b). MGRL medium (Fujiwara et al. 1992, Hirai et al. 1995) was used as a nutrient source and replaced with fresh medium every week. Phloem sap extraction Plants were grown hydroponically for 8 weeks in the same conditions as described above. Phloem sap was extracted from the petioles of rosette leaves as described in Tetyuk et al. (2013) with slight modifications such as Na2-EDTA was used instead of K2-EDTA and petioles were incubated in distilled water in the dark at 22°C for 8 h for phloem sap extraction. After the 8 h incubation, HCl was added at the final concentration of 0.01 N, frozen with liquid nitrogen and stored at –80°C until analysis of thiols and amino acids. Thiol analysis with HPLC Thiols extracted from plant tissues with 0.01 N HCl or phloem sap samples were derivatized with mono bromobimane, then cysteine, GSH, PC2, PC3 and PC4 were separated and quantified by HPLC, using standards (Minocha et al. 2008, Nishida et al. 2016). Standards for PC2, PC3 and PC4 were synthesized by Bonac Corporation (http://www.bonac.com). Amino acid analysis Amino acids extracted from tissues with 0.01 N HCl or phloem sap samples were derivatized with the AccQ-Fluor Reagent Kit (Waters, http://www.waters.com/waters/home) and measured with HPLC using an AccQ-Tag column (Waters) according to the manufacturer’s instructions. Analysis of Cd Plants were grown hydroponically in the same conditions as described above. Eight-week-old plants grown in normal MGRL medium were transferred to MGRL medium supplemented with 10 µM CdCl2 and grown for an additional 4 d. At the time of sampling, flowers were dried at 105°C for 24 h, weighed and then digested in 6 ml of a 5:1 (v/v) mixture of HNO3 and H2O2 in a microwave oven (ETHOS1600, Milestone). Analysis of Cd in digested solutions was performed with an inductively coupled plasma atomic emission spectrometer (IRIS Duo, Nippon Jarrell-Ash) as described in Nakamura et al. (2013, 2016). Observation of seeds with a stereo microscope After 8 weeks of hydroponic culture, young green siliques at approximately 2 cm were taken and opened by forceps. The seeds inside were observed using a stereo microscope (SZX16, Olympus) equipped with a DP25 camera (Olympus). Dried seeds were also observed by the same stereo microscope. Analysis of the time of bolting For comparison of the time of bolting, rockwool culture was used. Vernalization was performed by soaking seeds in distilled water for 3 d at 4°C. Seeds were sown on rockwool blocks (36 mm length, 36 mm width, 40 mm height, Grodan, http://www.grodan.com/) with one plant per block. Half-strength MGRL medium (Fujiwara et al. 1992, Hirai et al. 1995) or distilled water was applied alternately every 2 or 3 d to the rockwool height of 10 mm. The dates of bolting and numbers of rosette leaves at the time of flowering were recorded. Analysis of germination rates Plants were grown on rockwool as described above until their seeds were completely dried. The dried seeds of wild-type, GK-448H05 and GK-029H12 plants were harvested on the same day and stored in a desiccator at room temperature for about 3 months before being tested for germination. Harvested seeds were sterilized and sown on agar plates (Murashige and Skoog salts, 0.4 g l−1 MES, 20 g l−1 sucrose, pH 5.8, solidified with 10 g l−1 agar). 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Google Scholar CrossRef Search ADS PubMed  Zhang Z.C., Xie Q.Q., Jobe T.O., Kau A.R., Wang C., Li Y.X., et al.   ( 2016) Identification of AtOPT4 as a plant glutathione transporter. Mol. Plant  9: 481– 484. Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations Cd cadmium GCT γ-glutamyl transferase GSH glutathione OPT oligopeptide transporter PC phytochelatin RT–PCR reverse transcription–PCR © The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant and Cell Physiology Oxford University Press

AtOPT6 Protein Functions in Long-Distance Transport of Glutathione in Arabidopsis thaliana

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0032-0781
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Abstract

Abstract The involvement of the Arabidopsis oligopeptide transporter AtOPT6, which was previously shown to take up glutathione (GSH) when expressed in yeast cells or in Xenopus laevis oocytes, in GSH transport was analyzed using opt6 knockout mutant lines. The concentration of GSH in flowers or siliques was lower in opt6 mutants relative to wild-type plants, suggesting involvement of AtOPT6 in long-distance transport of GSH. The GSH concentration in phloem sap was similar between opt6 mutants and wild-type plants. These results, combined with earlier reports showing expression of AtOPT6 in the vascular bundle, especially in the cambial zone, suggest that AtOPT6 functions to transport GSH into cells surrounding the phloem in sink organs. The opt6 mutant plants showed delayed bolting, implying the importance of AtOPT6 for regulation of the transition from vegetative to reproductive growth. After cadmium (Cd) treatment, the concentration of the major phytochelatin PC2 was lower in flowers in the opt6 mutants and Cd was accumulated in roots of opt6 mutant plants compared with wild-type plants. These results suggest that AtOPT6 is likely to be involved in transporting GSH, PCs and Cd complexed with these thiols into sink organs. Introduction Glutathione is a sulfur-containing tripeptide comprised of l-γ-glutamyl-l-cysteinyl-glycine, which performs various important roles in plant cells such as redox homeostasis, detoxification of heavy metals and storage of organic sulfur (Noctor et al. 2011). Glutathione within the cell is primarily maintained in the reduced state (GSH), which is converted to the oxidized state (GSSG) in buffering reactive oxygen species, but in most cases is converted back to GSH by glutathione reductase. GSH is also involved in regulation of protein activity by changing protein thiol status (Foyer and Noctor 2005). Phytochelatins (PCs), the polymerized form of GSH, can detoxify heavy metals and metalloids such as cadmium (Cd) and arsenic (As) by chelating them (Cobbett 2000, Mendoza-Cózatl et al. 2011). GSH is also involved in regulation of plant growth, such as flowering (Ogawa et al. 2001, Yanagida et al. 2004, Hatano-Iwasaki and Ogawa 2007) and cell division (Vernoux et al. 2000, Vivancos et al. 2010a, Vivancos et al. 2010b). GSH functions as a major storage transport form of organic sulfur in plants. Sulfur is taken up from the environment into root cells mainly as sulfate, and transported into chloroplasts for reduction to sulfite then sulfide. Sulfide is assimilated into cysteine by reacting with O-acetyl-l-serine. However, plants primarily use GSH as a form of organic sulfur (Leustek et al. 2000). GSH is synthesized from the constituent amino acids in a two-step reaction by γ-glutamyl-cysteine synthetase (Hell and Bergmann 1990) and GSH synthetase (Wang and Oliver 1996), which are encoded by single genes GSH1 (May and Leaver 1994) and GSH2 (Wang and Oliver 1996), respectively. The evidence that GSH is the major form of organic sulfur stored and transported from sink to source organs was provided in experiments using Nicotiana tabacum fed with 35S-labeled sulfate (Rennenberg et al. 1979). Kuzuhara et al. (2000) observed that the GSH concentration in phloem sap taken from Oryza sativa using the insect laser method was around 5 mM, which is several times higher than the sulfate concentration, suggesting that GSH is the main form of sulfur transported through the phloem. GSH translocated through the phloem was also shown to be a signal to regulate sulfate uptake and ATP sulfurylase activity in Brassica napus (Lappartient and Touraine 1996). Although long-distance transport of GSH through the phloem is important for distribution of sulfur in plants, little is understood about the transporters involved in this process. AtMRP1 and AtMRP2 were identified as transporters for GSH and GSH conjugates into plant vacuoles, and AtCLTs were shown to transport GSH from chloroplasts into the cytosol (Lu et al. 1997, Lu et al. 1998, Maughan et al. 2010, Bachhawat et al. 2013). However, none of these transporters was implicated in the transport of GSH through the phloem. In Saccharomyces cerevisiae, Hgt1 was identified as a high-affinity GSH transporter (Km = 54 µM), and a yeast hgt1Δ mutant was unable to grow on a medium with GSH as a sulfur source (Bourbouloux et al. 2000). Hgt1 belongs to the oligopeptide transporter (OPT) family with 12–14 transmembrane domains. The Arabidopsis genome encodes nine possible OPT homologs, named AtOPT1–AtOPT9 (Koh et al. 2002). Expression of AtOPT1, 4, 5, 6 and 7 in yeast conferred the ability to take up tetra- and pentapeptides (Koh et al. 2002). Cagnac et al. (2004) observed that complementation of the yeast hgt1Δ mutant by AtOPT6 restored growth on a medium with GSH or GSSG as the sole sulfur source, and induced [3H]GSH transport into cells. Reporter gene experiments showed that AtOPT6 is mainly expressed in the vascular bundles of leaves, petioles and stems in plants (Cagnac et al. 2004, Stacey et al. 2006), suggesting involvement of AtOPT6 in GSH transport through the vascular tissue. In unfertilized gynoecium in flowers, expression of AtOPT6 was high in ovules, indicating a possible role for AtOPT6 in ovule development (Stacey et al. 2006). Pike et al. (2009) demonstrated that AtOPT6 expressed in Xenopus laevis oocytes transported GSH and PC2, but the Km for GSH was 566 µM, which was much higher than those for other peptides such as 10.8 µM for KLLLG and 71 µM for AtCLE19p12. Hence, the authors suggested that AtOPT6 may mediate the movement of signaling peptides. Recently, Zhang et al. (2016) reported that AtOPT4 complemented a yeast hgt1Δmet15Δ mutant for growth on a medium with GSH as the sole sulfur source and induced [35S]GSH transport into cells. The double knockout mutant opt2/opt4 in Arabidopsis showed a reduced content of GSH in siliques, suggesting a possible role for AtOPT4 in transporting GSH to siliques (Zhang et al. 2016). AtOPT3 was proposed to mediate shoot to root signaling of the Fe/Zn/Mn status for maintenance of these trace elements and it was also reported that misregulation of AtOPT3 led to an overaccumulation of cadmium in seeds (Stacey et al. 2008, Mendoza-Cózatl et al. 2014). A few OPT transporters have been studied in plants other than Arabidopsis. For example, BjGT1 from Brassica juncea (Bogs et al. 2003) and OsGT1 from Oryza sativa (Zhang et al. 2004) were also shown to complement a hgt1Δ mutant for growth with GSH as the sole sulfur source and induce [3H]GSH transport into cells. In the present study, we investigated the involvement of AtOPT6 in long-distance GSH transport in planta by analyzing the phenotype of Atopt6 mutant plants. Results Glutathione content in source and sink organs in the T-DNA insertional mutants of OPT6 In order to study the functions of AtOPT6 in planta, we obtained the T-DNA insertional mutant GK-029H12 which results from T-DNA insertion in the second exon of the AtOPT6 gene (At4g27730) (Supplementary Fig. S1A). We confirmed the lack of measureable OPT6 transcript in the homozygous GK-029H12 line by reverse transcription–PCR (RT–PCR; Supplementary Fig. S1B). To compare the distribution of GSH in whole plants, GK-029H12 and the corresponding wild-type (Columbia-0) plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) in normal MGRL hydroponic solution (Fujiwara et al. 1992, Hirai et al. 1995) followed by determination of the GSH content in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm in length) (Fig. 1A). There was no visible phenotype in the mutants compared with wild-type plants after 8 weeks of growth post-germination. The GSH content in flowers and siliques was decreased significantly in GK-029H12 mutant plants compared with wild-type plants, while there was no difference in GSH content in leaves and stems (Fig. 1A). This suggests that the GSH synthesis capacity in source leaves was not inhibited but translocation of GSH from source to sink organs was reduced in the mutant plants. Fig. 1 View largeDownload slide GSH concentration in the wild type (WT), and GK-029H12 and GK-448H05 mutants, and accumulation of AtOPT6 mRNA in WT plants. Plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark). GSH contents in roots, rosette leaves, cauline leaves, stems, flowers and siliques in GK-029H12 (A) or GK-448H05 (B) together with those in WT plants were determined. Means ± SDs of four biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). In (C), the levels of AtOPT6 mRNA in roots, rosette leaves, cauline leaves, stems, flowers and green siliques were determined in WT plants and normalized to those of ACTIN8. Means ± SDs of three biological replicates are shown, with the mean in roots set as 1. Fig. 1 View largeDownload slide GSH concentration in the wild type (WT), and GK-029H12 and GK-448H05 mutants, and accumulation of AtOPT6 mRNA in WT plants. Plants were hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark). GSH contents in roots, rosette leaves, cauline leaves, stems, flowers and siliques in GK-029H12 (A) or GK-448H05 (B) together with those in WT plants were determined. Means ± SDs of four biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). In (C), the levels of AtOPT6 mRNA in roots, rosette leaves, cauline leaves, stems, flowers and green siliques were determined in WT plants and normalized to those of ACTIN8. Means ± SDs of three biological replicates are shown, with the mean in roots set as 1. In order to confirm that the decreased GSH levels in sink organs were caused by the lack of OPT6 transcript, another T-DNA insertional mutant line, GK-448H05, was included in this analysis. The GK-448H05 mutant results from T-DNA insertion in the second exon of the AtOPT6 gene, and we confirmed the lack of measureable AtOPT6 transcript in the homozygous GK-448H05 line by RT–PCR (Supplementary Fig. S1C). As shown in Fig. 1B, GSH concentrations in sink organs such as siliques and flowers were significantly decreased in GK-448H05 compared with those in wild-type plants. Although absolute values of concentrations were different in Fig. 1A and B, which is probably due to difference in environmental conditions since the plants used for Fig. 1A and B were cultured independently, values of GSH of mutants relative to wild-type plants in flowers and siliques were comparable, 87% and 71% in GK-029H12 (Fig. 1A), and 61% and 73% in GK-448H05, respectively. The decrease in GSH levels in sink organs in the opt6 mutants indicates that OPT6 contributes the most in these organs. To confirm this, OPT6 gene expression was analyzed in various organs in wild-type plants. Total RNA was extracted from roots, rosette leaves, cauline leaves, stems, flowers and siliques of wild-type plants hydroponically grown for 8 weeks as above and OPT6 mRNA accumulation was determined by real-time PCR. As shown in Fig. 1C, expression of OPT6 in flowers and siliques was highest among the tissues examined, >3-fold higher than that in rosette leaves and >2-fold higher than that of cauline leaves. These expression patterns of OPT6 suggest that OPT6 protein contributes the most in sink organs, which is nicely correlated with the significant decrease in GSH concentration in flowers and siliques of the mutant plants (see Fig. 1A, B). Glutathione content in phloem sap of the opt6 mutant plants Previously published reports demonstrated that expression of AtOPT6 is mainly in vascular tissues (Cagnac et al. 2004, Stacey et al. 2006). Given these observations and the reduction of GSH translocation from source to sink organs in opt6 mutants (Fig. 1), we hypothesized that loading GSH into phloem was decreased by the mutations in the AtOPT6 gene. To confirm this, GSH contents in phloem sap in the GK-029H12 mutant were compared with those in wild-type plants. Plants were hydroponically grown for 8 weeks in the same way as used for GSH analysis above. To extract phloem sap, petioles were cut from rosette leaves, and were then soaked in EDTA solution for 1 h followed by soaking in distilled water for 8 h under darkness as described in Tetyuk et al. (2013). In order to normalize for the efficiency of extraction of phloem sap, GSH concentrations in the phloem sap were normalized to the glutamate concentration determined in the same samples. Previously, Cagnac et al. (2004) reported that GSH transport by AtOPT6 is inhibited by glutamine but not by glutamate, suggesting that AtOPT6 may transport glutamine but not glutamate, thus the concentration of glutamate was used for normalization. GSH concentrations before normalization and concentrations of major amino acids are presented in Supplementary Table S1. As noted, glutamate concentrations were not significantly different between mutants and the wild type. As shown in Fig. 2, the GSH contents in phloem sap normalized to glutamate content were not significantly different between GK-029H12 and wild-type plants. As a decrease of phloem GSH was not observed in the mutant, it is considered that OPT6 is not involved in loading of GSH into the phloem in source organs. Fig. 2 View largeDownload slide GSH concentrations normalized to glutamate concentrations in phloem sap from the wild type (WT) and GK-029H12 mutants. Phloem sap was extracted from rosette leaves of plants hydroponically grown for 8 weeks. GSH concentrations are normalized to glutamate concentrations. Values are (GSH concentrations/glutamate concentrations)×100. The means ± SDs of four biological replicates are shown. Fig. 2 View largeDownload slide GSH concentrations normalized to glutamate concentrations in phloem sap from the wild type (WT) and GK-029H12 mutants. Phloem sap was extracted from rosette leaves of plants hydroponically grown for 8 weeks. GSH concentrations are normalized to glutamate concentrations. Values are (GSH concentrations/glutamate concentrations)×100. The means ± SDs of four biological replicates are shown. The opt6 mutant plants show a delay in flowering Since GSH was reported to be involved in the transition from the vegetative rosette stage to the reproductive growth stage (Ogawa et al. 2001, Yanagida et al. 2004, Hatano-Iwasaki and Ogawa 2007), we examined the time of flowering in opt6 mutants relative to the wild type. GK-448H05, GK-029H12 and wild-type plants were grown hydroponically on rockwool under long-day conditions (16 h light/8 h dark). As shown in Fig. 3, bolting was delayed in both mutant lines compared with wild-type plants. The time at which >50% of total plants (n = 20) bolted was 30, 33 and 36 d after germination for the wild type, GK-448H05 and GK-029H12, respectively. Fig. 3 View largeDownload slide Timing of bolting in the wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were grown hydroponically on rockwool. The cumulative number of plants bolted from 27 to 39 d after germination was counted each day; n = 20. Fig. 3 View largeDownload slide Timing of bolting in the wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were grown hydroponically on rockwool. The cumulative number of plants bolted from 27 to 39 d after germination was counted each day; n = 20. The number of rosette leaves on bolting plants, which is an indicator of timing of bolting (Ogawa et al. 2001), was also counted. As shown in Table 1, both GK-448H05 and GK-029H12 mutant plants had an increased number of rosette leaves compared with wild-type plants, consistent with the delay in flowering shown by the mutant plants. A representative picture of the wild-type and opt6 mutants undergoing bolting is shown in Supplementary Fig. S2. Table 1 The number of rosette leaves on bolting plants   Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**    Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**  Values are means ± SD (n = 20). Asterisks indicate a significant difference at *P < 0.05 and *P < 0.01 (Student’s t-test), compared with the wild type. Table 1 The number of rosette leaves on bolting plants   Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**    Wild type  GK-448H05  GK-029H12  Rosette leaves  10.3 ± 1.7  11.4 ± 1.4*  13.4 ± 1.9**  Values are means ± SD (n = 20). Asterisks indicate a significant difference at *P < 0.05 and *P < 0.01 (Student’s t-test), compared with the wild type. The opt6 mutant seeds show lower germination rates Since expression of AtOPT6 is high in ovules (Stacey et al. 2006), AtOPT6 was suggested to be involved in seed development. To examine this, seeds in young and mature siliques were observed under a stereo microscope. Seeds of GK-448H05 and GK-029H12 mutant plants were normal and there was no apparent difference compared with wild-type seeds both in young green siliques and in mature dried siliques (Supplementary Fig. S3). To confirm proper maturity of seeds, the germination rate was compared between the opt6 mutants and wild-type plants. Twenty seeds per agar plate were sown in triplicate, and germination rates were calculated for each plate. As shown in Table 2, the germination rates of GK-448H05 and GK-029H12 seeds were less than half compared with that of wild-type plants. This suggests that lack of OPT6 inhibited proper seed maturation. Although germination rates were lower in the mutants, the rate of growth of the germinated plants was roughly equivalent to that of the wild type (data not shown). Table 2 The germination rates on agar plates   Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*    Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*  Values are means ± SD (n = 3). Asterisks indictates a significant difference at P < 0.01 (Student’s t-test) compared with the wild type. Table 2 The germination rates on agar plates   Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*    Wild type  GK-448H05  GK-029H12  Germination rate (%)  98.3 ± 2.9  40.0 ± 5.0*  30.0 ± 22.9*  Values are means ± SD (n = 3). Asterisks indictates a significant difference at P < 0.01 (Student’s t-test) compared with the wild type. Phytochelatin and Cd distribution in the opt6 mutants after Cd treatment Cd is thought to be transported through phloem complexed with GSH or PCs (Mendoza-Cózatl et al. 2011). Therefore, we also investigated involvement of OPT6 in Cd distribution using GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown under long-day conditions for 8 weeks, during which they were exposed to Cd for 4 d. GSH, PC2, PC3 and PC4 contents in source and sink organs were determined. No visible symptom of Cd stress was observed in any genotype at sampling time. As shown in Fig. 4B–D, PC2 content was higher than that of PC3 or PC4 in most organs, which suggests that PC2 is the major PC accumulated by Cd treatment in our conditions. The GSH concentration in rosette leaves was higher in opt6 mutants than that in wild-type plants (Fig. 4A). Concentrations of PC2 in flowers were significantly lower in both mutant lines compared with those in wild-type plants (Fig. 4B), suggesting that lack of AtOPT6 leads to a decrease in PC2. In source organs, such as rosette leaves, no differences in PC2 content were observed between the opt6 mutants and wild-type plants. PC3 and PC4 contents were not changed in rosette leaves, but in sink organs, such as siliques, PC4 levels were decreased in both mutant lines compared with wild-type plants. Fig. 4 View largeDownload slide GSH and PC concentrations in wild-type (WT), GK-448H05 and GK-029H12 mutant plants after Cd treatment. Plants hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) were treated with 10 μM Cd for 4 d. GSH (A), PC2 (B), PC3 (C) and PC4 (D) contents in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm) were determined. Means ± SDs of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05, *P < 0.1). Fig. 4 View largeDownload slide GSH and PC concentrations in wild-type (WT), GK-448H05 and GK-029H12 mutant plants after Cd treatment. Plants hydroponically grown for 8 weeks under long-day conditions (16 h light/8 h dark) were treated with 10 μM Cd for 4 d. GSH (A), PC2 (B), PC3 (C) and PC4 (D) contents in roots, rosette leaves, cauline leaves, stems, flowers and green siliques (1–2 cm) were determined. Means ± SDs of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05, *P < 0.1). If PCs are involved in Cd movement, then the distribution of Cd should reflect changes in PC content. Plants hydroponically grown and treated with Cd at the same time as those used for PC determination were subjected to Cd analysis by inductively coupled plasma atomic emission spectroscopy (ICP-AES). To obtain sufficient tissue for the measurement, flowers and siliques were pooled as sink tissues and used for a single Cd determination. As shown in Fig. 5, Cd content was highest in roots relative to other organs. In roots, Cd concentrations were >2-fold higher both in GK-448H05 and GK-029H12 compared with wild-type plants (Fig. 5A). These results suggest that lack of functional AtOPT6 caused accumulation of Cd in roots. Fig. 5 View largeDownload slide Cd concentration in wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown and treated with Cd at the same time as those used for Fig. 5. Cd concentrations in roots (A) cauline leaves, stems, flowers and green siliques (1–2 cm) (B) were determined. Means ± SD of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). Fig. 5 View largeDownload slide Cd concentration in wild-type (WT), GK-448H05 and GK-029H12 mutant plants. Plants were hydroponically grown and treated with Cd at the same time as those used for Fig. 5. Cd concentrations in roots (A) cauline leaves, stems, flowers and green siliques (1–2 cm) (B) were determined. Means ± SD of three biological replicates are shown. Asterisks indicate significant differences from the WT (Student’s t-test, **P < 0.05). Discussion GSH levels in sink organs such as flowers and siliques were decreased in opt6 mutants compared with wild-type plants (Fig. 1A, B). Absolute values of GSH content varied between Fig. 1A and B, which we attribute to different growth conditions since the plants used for Fig. 1A and B were grown independently. GSH concentrations are known to fluctuate depending on slight changes in environmental conditions. For example, GSH contents in shoots of Arabidopsis wild-type (Col-0) plants grown on normal MGRL agar plates for 10 d were about 500 nmol g FW−1 in Maruyama-Nakashita et al. (2006) and 250 nmol g FW−1 in Yamaguchi et al. (2016). In our analysis, the values of mutants and wild-type plants grown in the same culture were compared and the values of GSH of mutants relative to wild-type plants in flowers and siliques were similar in GK-029H12 and GK-448H05. This suggests that AtOPT6 is involved in long-distance transport of GSH from source to sink organs. As GSH is the major transport form of sulfur (Rennenberg et al. 1979), AtOPT6 may have an important role in distribution of sulfur in plants. Transport of GSH to sink organs was not completely inhibited in opt6 mutants, implying that there may be other routes for GSH transport. AtOPT2 and AtOPT4 are likely candidates since opt2/opt4 double mutant plants showed reduced GSH concentration in siliques compared with wild-type plants (Zhang et al. 2016). AtOPT6 is mainly expressed in plant vascular tissues (Cagnac et al. 2004, Stacey et al. 2006). Hence, we hypothesized that transport of GSH through the phloem from source to sink tissues was decreased in opt6 mutants. However, the GSH content in phloem sap was not changed in opt6 mutant plants. This suggests that loading of GSH into the phloem is not inhibited in opt6 mutant plants (Fig. 2). Cagnac et al. (2004) observed in reporter gene experiments that expression of AtOPT6 was high in the cambial zone of the vascular bundles in the petiole and stem of adult plants. The cambial zone between the phloem and xylem consists of meristematic cells where nutrients are required for actively dividing cells. After unloading from phloem companion cells in sink organs, solutes are transported into the apoplast and then incorporated into cells surrounding the phloem. We propose that AtOPT6 is the transporter for incorporating GSH into cells surrounding the phloem such as cells in the cambial zone from the apoplastic space. In our experiments, OPT6 mRNA expression was higher in sink organs such as flowers and siliques compared with leaves, stems and roots (Fig. 1C), which is in agreement with the findings of Koh et al. (2002) and also supports the idea that AtOPT6 functions in absorbing GSH into cells in sink organs transported through the phloem from source organs. The time of bolting for opt6 mutant plants was delayed (Fig. 3; Table 1), suggesting that AtOPT6 contributes to the transition from vegetative to reproductive growth. Two possible mechanisms may explain the contribution of AtOPT6 to flowering; one is through transportation of GSH and another is through transportation of other peptides. The Arabidopsis cad-1 mutant has reduced amounts of GSH due to a mutation in the γ-glutamyl-cysteine synthetase gene and also displays delayed flowering. This phenotype is stronger when an inhibitor of γ-glutamyl-cysteine synthetase is added and is abolished by application of GSH (Ogawa et al. 2001). In Eustoma grandiflorum, induction of bolting by vernalization was nullified by inhibition of GSH synthesis, while addition of GSH induced bolting (Yanagida et al. 2004). Hatano-Iwasaki and Ogawa (2007) suggested that a crucial amount of GSH synthesized during early growth is important for promotion of flowering. Our results, together with those of previous studies, suggest the possibility that GSH incorporated into sink organs by AtOPT6 is involved in regulation of flowering time. Alternatively, we cannot exclude the possibility that transport of other, unidentified peptides by AtOPT6 contributes to flowering time regulation. Pike et al. (2009) reported that AtOPT6 expressed in X. laevis oocytes transported not only GSH but also other peptides, including some CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) peptides that are known to be mobile signals controlling cell differentiation and plant development. It is possible that the delay in bolting is influencing the amount of GSH and PC in flowers and siliques. However, we view this as less likely because a special effort was made to sample flowers and siliques at identical developmental stages, opened flowers and green siliques 1–2 cm in length, respectively. The seed germination rate was reduced more than half in both mutant lines compared with the wild type (Table 2), indicating that seed development was inhibited by the lack of AtOPT6. Although the appearance of seeds from the wild-type and mutant plants was not different (Supplementary Fig. S3), it is likely that their metabolism differs. Cairns et al. (2006) showed that a functional GSH1 was required for maturation of Arabidopsis seeds. Our results coupled with the report of Cairns et al. (2006) suggest that GSH is a key metabolite contributing to seed maturation. Stacey et al. (2006) observed that expression of AtOPT6 was high in ovules in unfertilized gynoecium in flowers, thus it is possible that AtOPT6 contributes not only to seed maturation but also to development of ovules. Recently Giaretta et al. (2017) demonstrated the roles of the γ-glutamyl transferases GGT1 and GGT2 in delivery of cysteine in seeds. GGT1 and GGT2 are both located in the apoplast and involved in GSH degradation (Ohkama-Ohtsu et al. 2007a). Giaretta et al. (2017) suggested that GSH transported from leaves to seeds through the phloem followed by unloading is released to the apoplast of the outer and inner integument, where GGT1 is strongly expressed and may assist in GSH degradation to provide cysteine to the endosperm. GGT2 expression is high in the suspensor and micropylar endosperm during embryo development and may function in supplying cysteine to the embryo from the micropylar endosperm. In their study, silencing of both ggt1 and ggt2 by RNA interference resulted in low seed yields. They concluded that the extracellular GGTs integrate sulfur levels when demand is high by degrading GSH. It is possible that OPT6 and GGTs co-ordinately function in delivery of sulfur into seeds, where OPT6 takes up GSH unloaded from the phloem into cells of ovules followed by releasing it into apoplast of the outer and inner integument, and degradation into cysteine by GGT1 and GGT2. In opt6 mutant plants, PC content after Cd treatments was also lower in flowers compared with wild-type plants (Fig. 4). PC2 was the major PC accumulated, relative to PC3 or PC4. The concentration of PC2 in flowers was significantly lower in both opt6 mutants compared with wild-type plants (Fig. 4B). Although not as significant, the content of PC3 and PC4 also tended to be lower in opt6 mutants (Fig. 4C, D). Pike et al. (2009) reported that AtOPT6 expressed in X. laevis oocytes allowed transport of PC2. Thus, we propose that AtOPT6 transports PCs in addition to GSH, probably into cells surrounding the phloem in sink organs. Cd accumulated in roots in both GK-448H05 and GK-029H12 mutant plants, relative to wild-type plants (Fig. 5A). Cd is thought to be transported in the phloem complexed with GSH or PCs (Mendoza-Cózatl et al. 2011). Cagnac et al. (2004) showed that yeast hgt1Δ mutant cells complemented with AtOPT6 had the ability to transport GS conjugates. They also observed that complementation with AtOPT6 increased yeast sensitivity to Cd. This sensitivity was enhanced when GSH was added to the medium. The authors suggested that AtOPT6 transports Cd conjugated with GSH (Cagnac et al. 2004). As expression of OPT6 is high in flowers compared with other organs (Fig. 1C), it is possible that AtOPT6 functions in unloading of GSH–Cd conjugates and PC–Cd conjugates from the phloem into flowers and siliques. Mendoza-Cózatl et al. (2011) suggested that GSH–Cd conjugates and PC–Cd conjugates loaded into the phloem are more likely to be sequestered in root vacuoles by the PC transporters ABCC1 and ABCC2, which is supported by measurements of the transcript levels of ABCC1 and ABCC2, which are on average 3-fold higher in roots compared with those in shoots (Mustroph et al. 2009). It is possible that inhibition of unloading into flowers and siliques in the opt6 mutants further enhanced the movement of thiol–Cd complexes in phloem towards roots for sequestering into the root vacuoles, which caused Cd accumulation in roots in the mutants (Fig. 5A). Although unloading of thiol–Cd complexes appears to be inhibited in the mutants, the concentration of Cd in flowers and siliques was not changed in GK-448H05 and was only slightly higher in GK-029H12 compared with wild-type plants. This suggests that another route probably exists for Cd entering into flowers independently of AtOPT6. However, in general, flowers and siliques are not major sinks for Cd given the very low concentrations measured in these organs. Recently another gene in the OPT family in Arabidopsis, AtOPT3, was shown to be involved in Cd distribution in plants (Mendoza-Cózatl et al. 2014). The opt3 mutant plants accumulated Cd in roots and seeds, and underaccumulated Cd in leaves, while they accumulated Fe and Zn in both roots and leaves compared with wild-type plants. AtOPT3 was suggested to function in signaling of Fe status from shoot to roots. However, the role of AtOPT3 in Cd movement may be independent of Fe signaling because the pattern of Fe distribution in the opt3 mutant was different from that of Cd (Stacey et al. 2008, Mendoza-Cózatl et al. 2014). These previous studies with AtOPT3 and our results with AtOPT6 demonstrated that transporters in the OPT family play key roles in Cd movement in plants. In conclusion, our present study together with those of Koh et al. (2002), Cagnac et al. (2004), Stacey et al. (2006) and Pike et al. (2009) suggest that AtOPT6 transports GSH, and PCs and Cd complexed with these thiols into actively dividing cells around phloem in sink organs. In this way, AtOPT6 contributes to the distribution of organic sulfur and GSH incorporated into sink organs, which in turn is critical for maintenance of redox homeostasis of cells, as well as for the myriad of reactions that require organic sulfur. However, as indicated by Cagnac et al. (2004) and Pike et al. (2009), AtOPT6 appears to have broad specificity with regard to peptide transport and, hence, may play additional roles beyond the movement of GSH and PCs in planta. Materials and Methods Plant materials The GK-029H12 mutant (ecotype Columbia-0) was provided by GABI-kat (http://www.gabi-kat.de/) (Rosso et al. 2003). The GK-029H12 line is a result of a T-DNA insertion in the second exon of the AtOPT6 gene. The homozygous plants were screened by PCR using the gene-specific primers GK-029-F (5'-CTCATGCCTCCTCTTGATTTC-3') and GK-029-R (5'-CTTCAACGTCAAGGAACATG-3'), and the T-DNA left-border primer GABI-8474 (5'-ATAATAACGCTGCGGACATCTACATTTT-3') (http://www.gabi-kat.de/). A lack of amplifiable transcript of AtOPT6 in the GK-029H12 mutant was confirmed by quantitative RT–PCR using primers OPT6RT-F (5'-GAAGAGCTGACTAGAGAAGATAGG-3') and OPT6RT-R (5'-ACCACTCAGATTCTTGGATTTG-3'). The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). The GK-448H05 mutant (ecotype Columbia-0) was provided by GABI-kat (http://www.gabi-kat.de/) (Rosso et al. 2003). The GK-448H05 line is a result of a T-DNA insertion in the second exon of the AtOPT6 gene. The homozygous plants were screened by PCR using the gene-specific primers GK-448-F (5'-AACATGATAAGCGCGATA-3') and GK-448-R (5'-AGTCTCTCTCTTTGGTGCGTTACA-3'), and the T-DNA left-border primer GABI-8474 (http://www.gabi-kat.de/). A lack of amplifiable transcript of AtOPT6 in the GK-448H05 mutant was confirmed by quantitative RT–PCR using primers OPT6RT-F and OPT6RT-R described above. The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). RT–PCR Total RNA was treated with DNase I (Thermo Fisher Scientific, https://www.thermofisher.com/us/en/home.html) and used as a template to synthesize cDNA with SuperScrpt III reverse transcriptase (Thermo Fisher Scientific) and oligo(dT)20 according to the manufacturer’s instructions. PCR analysis was performed with KOD plus NEO (TOYOBO Life Science, http://lifescience.toyobo.co.jp). Quantitative real-time PCR analysis Real-time PCR analysis was performed with LightCycler® Nano (Roche, https://lifescience.roche.com/shop/home) and Essential DNA Green Master (Roche) as recommended by the manufacturer. The primers used for quantifying the OPT6 transcripts were 5'-TCAACGGAAGTGACTTGTGG-3' and 5'-ACCACCACAATGGAACTTCC-3'. The ACTIN8 gene was chosen as a control, and its expression was determined using primers as described by Goto and Naito (2002). Hydroponic culture of plants Unless otherwise indicated, Arabidopsis thaliana plants were grown hydroponically for 8 weeks in 50 ml conical tubes at 22°C under 16 h light (150 μmol m−2 s−1)/8 h dark as described in Ohkama-Ohtsu et al (2007b). MGRL medium (Fujiwara et al. 1992, Hirai et al. 1995) was used as a nutrient source and replaced with fresh medium every week. Phloem sap extraction Plants were grown hydroponically for 8 weeks in the same conditions as described above. Phloem sap was extracted from the petioles of rosette leaves as described in Tetyuk et al. (2013) with slight modifications such as Na2-EDTA was used instead of K2-EDTA and petioles were incubated in distilled water in the dark at 22°C for 8 h for phloem sap extraction. After the 8 h incubation, HCl was added at the final concentration of 0.01 N, frozen with liquid nitrogen and stored at –80°C until analysis of thiols and amino acids. Thiol analysis with HPLC Thiols extracted from plant tissues with 0.01 N HCl or phloem sap samples were derivatized with mono bromobimane, then cysteine, GSH, PC2, PC3 and PC4 were separated and quantified by HPLC, using standards (Minocha et al. 2008, Nishida et al. 2016). Standards for PC2, PC3 and PC4 were synthesized by Bonac Corporation (http://www.bonac.com). Amino acid analysis Amino acids extracted from tissues with 0.01 N HCl or phloem sap samples were derivatized with the AccQ-Fluor Reagent Kit (Waters, http://www.waters.com/waters/home) and measured with HPLC using an AccQ-Tag column (Waters) according to the manufacturer’s instructions. Analysis of Cd Plants were grown hydroponically in the same conditions as described above. Eight-week-old plants grown in normal MGRL medium were transferred to MGRL medium supplemented with 10 µM CdCl2 and grown for an additional 4 d. At the time of sampling, flowers were dried at 105°C for 24 h, weighed and then digested in 6 ml of a 5:1 (v/v) mixture of HNO3 and H2O2 in a microwave oven (ETHOS1600, Milestone). Analysis of Cd in digested solutions was performed with an inductively coupled plasma atomic emission spectrometer (IRIS Duo, Nippon Jarrell-Ash) as described in Nakamura et al. (2013, 2016). Observation of seeds with a stereo microscope After 8 weeks of hydroponic culture, young green siliques at approximately 2 cm were taken and opened by forceps. The seeds inside were observed using a stereo microscope (SZX16, Olympus) equipped with a DP25 camera (Olympus). Dried seeds were also observed by the same stereo microscope. Analysis of the time of bolting For comparison of the time of bolting, rockwool culture was used. Vernalization was performed by soaking seeds in distilled water for 3 d at 4°C. Seeds were sown on rockwool blocks (36 mm length, 36 mm width, 40 mm height, Grodan, http://www.grodan.com/) with one plant per block. Half-strength MGRL medium (Fujiwara et al. 1992, Hirai et al. 1995) or distilled water was applied alternately every 2 or 3 d to the rockwool height of 10 mm. The dates of bolting and numbers of rosette leaves at the time of flowering were recorded. Analysis of germination rates Plants were grown on rockwool as described above until their seeds were completely dried. The dried seeds of wild-type, GK-448H05 and GK-029H12 plants were harvested on the same day and stored in a desiccator at room temperature for about 3 months before being tested for germination. Harvested seeds were sterilized and sown on agar plates (Murashige and Skoog salts, 0.4 g l−1 MES, 20 g l−1 sucrose, pH 5.8, solidified with 10 g l−1 agar). 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Plant and Cell PhysiologyOxford University Press

Published: Apr 14, 2018

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