Members of the ATP Binding Cassette B/Multidrug-Resistance/P–glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4–D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (–Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition.
Auxin regulates many aspects of plant growth and development (Woodward and Bartel, 2005; Leyser, 2006; Teale et al., 2006). Auxin is thought to be synthesized in young apical tissues and then to enter basal tissues including the maturing stem and the roots by a polar transport system. The distribution of auxin and the formation of auxin gradients in the tissues are supposed to be directed by the activity of members of three families: the PIN-FORMED (PIN) family, AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) family and the ATP Binding Cassette B (ABCB) subfamily (Cho and Cho, 2012). Members of the long-looped PIN family seem to be responsible for the auxin efflux from cells (Krecek et al., 2009), whereas the AUX/LAX family charge the auxin influx into cells (Péret et al., 2012), and the auxin transport directionality of the ABCB subfamily seems to depend on the cytoplasmic auxin concentration (Yang and Murphy, 2009; Abel and Theologis, 2010; Peer et al., 2011).
The ABCB subfamily is the second largest ATP-binding cassette (ABC) protein subfamily and the largest full-molecule ABC transporter subfamily in plants (Rea, 2007). The function of ABCB in plants was first reported in the model plant Arabidopsis thaliana (Sidler et al., 1998). For now, five ABCB genes in Arabidopsis (AtABCB1, AtABCB4, AtABCB14, AtABCB19 and AtABCB21) have been functionally characterized. AtABCB1 and AtABCB19 (a close homolog of AtABCB1) were demonstrated to function as auxin efflux transporters (Sidler et al., 1998; Noh et al., 2001; Geisler et al., 2003, 2005; Cho et al., 2007; Wang et al., 2013). AtABCB4 and AtABCB21 (a close homolog of AtABCB4) were reported to have facultative auxin transport function, depending on auxin concentrations: demonstrating auxin uptake at low auxin concentrations, reversing to auxin export at high auxin concentrations (Terasaka et al., 2005; Yang and Murphy, 2009; Kamimoto et al., 2012; Kubeš et al., 2012). Additionally, AtABCB14 is a malate uptake transporter and plays an essential role under stress conditions (Lee et al., 2008). Studies of ABCBs in other species showed that ABCBs are also able to transport secondary metabolites (Shitan et al., 2003), as well as regulate aluminum tolerance and calcium homeostasis (Sasaki et al., 2002).
In Arabidopsis, evidence has been provided that the members of the PIN and the ABCB protein families regulate auxin transport coordinately and independently (Noh et al., 2003; Bandyopadhyay et al., 2007; Blakeslee et al., 2007; Mravec et al., 2008; Titapiwatanakun et al., 2009). Some studies also focused on the interaction between ABCBs and the immunophilin-like FKBP42, TWISTED DWARF1 (TWD1), and the protein phosphorylation of ABCBs. AtABCB1 was reported to be a substrate of PINOID (PID), an AGC kinase. TWD1 interacts with PID, and directs the phosphorylation of ABCB1 in a regulatory linker domain that alters ABCB1-mediated auxin transport activity (Geisler et al., 2003, 2004; Bouchard et al., 2006; Bailly et al., 2008; Henrichs et al., 2012). AtABCB19 can also be phosphorylated by PHOTOTROPIN 1 (phot1), a plasma membrane serine-threonine protein kinase involved in blue-light responses, to inhibit its efflux activity (Christie et al., 2011). The results described above were mainly established using dicotyledonous Arabidopsis as a model system, but the role of ABCBs is unclear in monocotyledons.
Several studies indicate a close interrelationship between auxin and iron (Fe) ion homeostasis: auxin has already been reported, since the 1980s, to be implicated in Fe deficiency-induced adaptive responses (Landsberg, 1981). Helianthus annuus (sunflower) roots were shown to have higher levels of auxin under Fe-deficient conditions than under Fe-sufficient conditions (Römheld and Marschner, 1986). Reductions of auxin transport or auxin sensitivity all inhibited the formation of root hair in response to Fe deficiency (Schikora and Schmidt, 2001). Furthermore, the influence of plant hormones on Fe uptake by strategy–I plants was summarized by Romera et al. (2007). Based on reports from recent years, Fe deficiency can trigger an over-accumulation of indole-3-acetic acid (IAA) and Arabidopsis auxin transporter aux1–7 mutant plants exhibit reduced ferric chelate reductase activity under low Fe (Chen et al., 2010). Thus, the auxin transporter AtAUX1 is thought to mainly direct the root-ward auxin stream into lateral roots to integrate local Fe nutritional status (Giehl et al., 2012). In Malus xiaojinensis, it is also indicated that Fe deficiency-induced physiological responses are mediated by systemic auxin signaling (Wu et al., 2012). We also reported that auxin response factor, OsARF12, regulates root elongation and affects Fe homeostasis in Oryza sativa (rice; Qi et al., 2012); however, the molecular mechanism of auxin in the regulation of Fe-deficiency responses remains unclear in monocots, and must be systematically investigated further.
It is important to note that, although rice is an important food crop, there are no reports on its ABCBs. To test whether ABCB also plays an important role in rice, we investigated the behavior of the OsABCB14 gene in rice. OsABCB14 is shown to function in cellular auxin uptake and Fe homeostasis in rice.
Identification of osabcb14 mutants and complemented transgenic lines
We obtained two TOS17 insertional lines in the OsABCB14 gene from the Rice Genome Resource Center in Japan. Using PCR analysis and sequencing, we confirmed that TOS17 had been inserted into the eighth exon of the OsABCB14 gene at osabcb14–1, and the seventh intron of OsABCB14 in osabcb14–2, respectively (Figure 1a). Both mutant lines are homozygous (Figure 1b). In addition, we overexpressed OsABCB14 in osabcb14–1 and osabcb14–2 under the control of a CaMV 35S promoter to create the complemented transgenic lines. Reverse transcriptase real-time PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) using three pairs of primers (located around the insertion sites, and the 5′ and 3′ regions, respectively) showed that the expression of OsABCB14 was about fivefold lower in both mutant lines and about fivefold higher in both complementation lines (osabcb14-1C and osabcb14-2C) than in the wild type (Nipponbare, NIP; Figure 1c,d).
AtABCB1 and AtABCB19, the two closest relatives of OsABCB14 (Shen et al., 2010), were reported to transport auxin. This led us to ask whether OsABCB14 was involved in auxin transport. We first investigated the phenotypes of shoots and primary roots (PRs) of NIP, osabcb14 mutants and osabcb14C. The shoot and PR lengths in NIP for 7–day-old seedlings were only slightly shorter than those of osabcb14–1 and osabcb14–2 under normal conditions (CK; Figure 1e), whereas they were significantly shorter than those of osabcb14–1 and osabcb14–2 under treatment with 0.01 μm 2,4–dichlorophenoxyacetic acid (2,4–D; Figure 1f). Similar results were obtained when grown in the nutritional solution containing 10 μm IAA (Figure 1g); however, there was no difference between NIP and osabcb14 mutants under treatment with 0.001–1 μm naphthylacetic acid (NAA; Figure S1). We further performed a dose–response assay to confirm the effect of 2,4–D, IAA and NAA in more detail. High concentrations of all three auxins inhibited shoot and PR growth, but this effects was more prominent in NIP than in osabcb14 mutants under 2,4–D treatment at a concentration of >10−8 mol L−1 (Figure 1h,i) and IAA treatment at a concentration of >10−7 mol L−1 (Figure 1j,k), whereas there was no significant difference under NAA treatment (Figure S1). All these phenotypes in osabcb14 mutants could be reverted by transgenic lines osabcb14C (Figure 1e–k). These results confirmed that osabcb14 mutants are insensitive to 2,4–D and IAA, but are not responsive to NAA. These observations were similar to the previous studies on AUX1 (Delbarre et al., 1996; Yang et al., 2006), suggesting that OsABCB14 is required for auxin transport in either shoot or root.
Expression pattern and subcellular localization of OsABCB14
To explore the in vivo function of OsABCB14, we first analyzed the expression pattern of OsABCB14. The expression pattern of OsABCB14 was analyzed in different organs and growth stages using qRT-PCR. The results showed that OsABCB14 expression is constitutive in various organs. At all growth stages observed, OsABCB14 was ubiquitously expressed in all plant organs, including the root, stem, leaf, node, root–stem transition region, filling seed, panicle and flower (Figure 2a). Spatial expression analysis showed that the expression of OsABCB14 was higher in root tips than in the basal root zones (Figure 2b). GUS staining revealed that OsABCB14 is expressed in the root tip and stele of the PR and leaves. A cross section of leaf sheath showed that OsABCB14 is expressed in the Vasculature (Figure 2c–f). OsABCB14 is also strongly expressed in the node, internode, root–stem transition region and flowers (Figure 2g–j).
To investigate the relationship between OsABCB14 and phytohormones, including auxin, the expression of OsABCB14 under treatment with various phytohormones and polar auxin transport inhibitors was analyzed by qRT-PCR. As shown in Figure 2k and Figure S2a, the expression of OsABCB14 was greatly induced by treatment with 2,4–D, IAA, 6–benzyladenine, abscisic acid, salicylic acid or naphthoxyacetic acid, the expression of OsABCB14 was only slightly upregulated by treatment with NAA, GA3 and NPA. Furthermore, when treated with 1 μm 2,4–D or 10 μm IAA, the expression of OsABCB14 peaked after 12 h (Figure 2l,m; Figure S2b,c).
To determine the subcellular localization of the OsABCB14 protein, OsABCB14 was cloned into pH7FWG2 and then fused with enhanced green fluorescent protein (EGFP). We observed transient expression of OsABCB14 in the epidermis cells of Nicotiana benthamiana (tobacco) leaves and Allium cepa (onion) epidermis, indicating that OsABCB14 was localized at the plasma membrane (Figure 3a–c).
osabcb14–1 and osabcb14–2 mutants own reduced auxin concentrations
The loss-of-function mutation of AtAUX1, which is an auxin influx transporter, resulted in reduced auxin concentration and auxin transport (Yu and Wen, 2013). In addition, the loss of mutations of AtABCB19, identified as auxin efflux transporters, also led to reduced auxin concentration and auxin transport (Noh et al., 2001). To determine whether the knock-down of OsABCB14 also alters auxin concentration and auxin transport in rice, we measured the auxin concentration and polar auxin transport in NIP and osabcb14 mutants. The results showed that the auxin concentrations of osabcb14–1 and osabcb14–2 in both shoot and root were significantly lower than in NIP (Figure 4a,b). The staining of DR5:GUS, a biological marker to study the native auxin distribution, was observed in NIP and osabcb14 mutants (Figure 4c,d), and the results coincided with the measurements described above. In addition, an examination of the transcription of well-characterized auxin-responsive genes in osabcb14 mutants showed that mRNA levels of OsIAA3, OsIAA9, OsIAA23 and OsSAUR39 were downregulated in osabcb14 mutants (Figure 4e,f), which is hence consistent with the reduced auxin sensitivity and auxin concentration in osabcb14 mutants.
Auxin transport activity in yeast and osabcb14 mutants
Reduced auxin sensitivities and auxin levels prompted us to quantify auxin transport capacities of OsABCB14. First, we functionally expressed OsABCB14 in Saccharomyces cerevisiae (baker's yeast). Yeast expressing OsABCB14 accumulated three times more IAA than the vector control, indicating auxin uptake activity for OsABCB14 (Figure 5a). Auxin uptake by OsABCB14 was specific as the diffusion control benzoic acid (BA) was not transported differently to the vector control. Next, we quantified auxin loading (i.e. influx) into rice protoplasts not yet shown before. Both osabcb14 alleles revealed significantly reduced auxin loading, pointing again to an auxin import activity of OsABCB14 (Figure 5b). In order to test the facultative efflux capacity of OsABCB14 reported for Arabidopsis orthologs AtABCB4 and AtABCB21 (Terasaka et al., 2005; Yang and Murphy, 2009; Kamimoto et al., 2012; Kubeš et al., 2012), we also quantified IAA efflux after the loading of protoplasts. Interestingly, both osabcb14 alleles revealed significantly enhanced IAA export, again indicating a preferred import directionality for OsABCB14, which, when absent, reduces efflux most probably by a lack of re-import of effluxed IAA (Figure 5c). In analogy to auxin treatments, no import of NAA by OsABCB14 was found to be independent of auxin uptake systems, as it can freely diffuse into cells (Delbarre et al., 1996; Yang et al., 2006). Finally, acropetal auxin transport of osabcb14–1 and osabcb14–2 roots was found to be decreased by ~59 and by ~63%, respectively, in comparison with NIP (Figure 5d), whereas basipetal auxin transport did not differ from NIP (Figure 5e). In summary, This suggests that OsABCB14 functions as an auxin importer involved in acropetal transport, but not basipetal transport, of auxin.
osabcb14 mutants are insensitive to Fe deficiency and OsABCB14 was involved in Fe homeostasis
TaABCB1 was reported to be involved in aluminum tolerance and calcium homeostasis in Triticum aestivum L. (wheat; Sasaki et al., 2002). To test whether OsABCB14 is also functionally related to ion homeostasis, we measured the metal ion concentrations in the seeds, shoots and roots of NIP and osabcb14 mutants. As shown in Figure 6a, the Fe concentrations in the seeds, shoots and roots of the two osabcb14 mutant lines were all significantly higher (56% higher in osabcb14–1 and 43% higher in osabcb14–2 for seeds; 51% higher in osabcb14–1 and 38% higher in osabcb14–2 for shoots; 35% higher in osabcb14–1 and 40% higher in osabcb14–2 for roots) than that in NIP. The concentrations of the other metals, such as Mn, Cu, Zn and Mg, were also enhanced in the osabcb14 mutants compared with NIP (Figure S3). These results suggested that OsABCB14 might be involved in ion homeostasis.
To understand the relationship between OsABCB14 and Fe homeostasis, we further investigated the phenotypes of osabcb14 mutants under Fe-deficient nutrient solution (–Fe). osabcb14 mutants displayed significantly longer shoots and roots than NIP under –Fe conditions, but only slightly longer roots than NIP and normal shoot length under CK (Figure 6b–d). The chlorophyll concentration of osabcb14 mutants was significantly higher (161% in osabcb14–1 and 183% in osabcb14–2) than that of NIP under –Fe (Figure 6e). In addition, the differences of Fe concentration between NIP and osabcb14 mutants were more obvious under –Fe treatment (65 and 40% higher in shoots and roots of osabcb14–1, 40 and 43% higher in shoots and roots of osabcb14–2; Figure 6f,g) compared with CK (Figure 6a). These results suggest that osabcb14 mutants are insensitive to –Fe.
The enhanced Fe concentrations in both shoots and roots of osabcb14 mutants under CK and –Fe may have resulted from enhanced Fe concentration in the seeds or impaired Fe uptake or homeostasis, or from both of these causes. To evaluate the effect of OsABCB14 disruption on the rice Fe uptake and signaling system, we analyzed the expression level of Fe deficiency-responsive genes IRT1 (Fe-regulated transporter 1), IRT2, IRO2 (Fe-responsive operator 2), YSL15 (yellow-stripe like 15), NAAT1 (nicotianamine aminotransferase 1), NRAMP2 (natural resistance-associated macrophage protein 2), NAS1 (nicotianamine synthase) and NAS2. The results showed that the expression of all these genes was upregulated in osabcb14 mutants (Figures 7a, S4a). These genes were reported to be upregulated under –Fe (Jia et al., 2011; Qi et al., 2012). Consistent with this finding, qRT-PCR analysis here showed that all these genes were induced in NIP and osabcb14 mutants under –Fe, but that the induced levels in osabcb14 mutants were significantly lower than in NIP (Figures 7b, S4b). This result closely coincided with the observations above: osabcb14 mutants are insensitive to Fe deficiency.
In the rice genome, 22 ABCB genes have been identified (Shen et al., 2010), but not one of them has been functionally characterized. Here, we report the biological function of OsABCB14 in auxin uptake and Fe homeostasis in rice.
Evidence presented in this and other articles demonstrates that many ABCB family members play critical roles in auxin-dependent development processes. For example, research on AtABCB1 and AtABCB19, the overexpression and disruption of which led to the deregulation of root elongation, gravitropism and phototropism (Noh et al., 2003; Lewis et al., 2007; Wu et al., 2007), suggests that they are involved in auxin-dependent programs. Several studies also supported the role for AtABCB4 in auxin-mediated developmental processes, mainly in root elongation (Santelia et al., 2005; Terasaka et al., 2005). OsABCB14, as the closest rice ortholog of AtABCB1 and AtABCB19, conferred altered sensitivity to high concentrations of 2,4–D and IAA (Figure 1e–k), suggesting its involvement in auxin-mediated programming.
The reduced responses of osabcb14–1 and osabcb14–2 to 2,4–D and IAA are similar to the previous studies on AtAUX1 (Yang et al., 2006). As a synthetic auxin that is transported into plant cells predominantly by a carrier (Delbarre et al., 1996; Yamamoto and Yamamoto, 1998), 2,4–D is very useful for assessing the activity of auxin uptake carriers (Hoyerová et al., 2008). A defect in the auxin uptake carrier is expected to lead to a decreased sensitivity to high concentrations of 2,4–D, and both osabcb14 mutants exhibited such a phenotype, suggesting that OsABCB14 may play an important role in the influx phase of polar auxin transport. These data are supported by the finding that osabcb14 shows an unaltered sensitivity to the synthetic auxin NAA (Figure S1), known to be independent of an uptake system, such as AUX1 (Delbarre et al., 1996; Yamamoto and Yamamoto, 1998) or ABCB4-like ABCBs (Kamimoto et al., 2012).
Results of IAA transport analysis from yeast and leaf protoplasts prepared from knock-down rice mutant alleles verify that OsABCB14 functions as an auxin transporter, and are in complete accordance with an uptake or import function (Figure 5). This finding is slightly surprising because OsABCB14 clusters closer to Arabidopsis ABCB19 (and ABCB1) than to ABCB4/ABCB21, which were characterized as an exporter and facultative importers/exporters, respectively. However, our data (especially efflux experiments from rice protoplasts; Figure 5c) indicate that OsABCB14 functions under tested conditions primarily as an importer. Therefore, these data also suggest that transport directionalities cannot simply be deduced by phylogenetic analyses, or that transport directionalities are achieved differently in monocots and dicots.
Acropetal auxin transport of both osabcb14 mutant roots was significantly lower than in NIP, suggesting that OsABCB14 functions in acropetal (root-ward) polar auxin transport. As a result, knock-down roots of OsABCB14 displayed reduced auxin concentrations and DR5:GUS activity (Figure 4a–d), most probably caused by reduced auxin delivery from the shoot. Interestingly, shoot IAA levels were also strongly reduced in both mutant alleles, suggesting altered shoot transport; however, we failed to quantify shoot transport in osabcb14 alleles reliably because measurements of shoot transport are still a technical challenge in rice. We speculate that, as in roots, acropetal (i.e. shoot-ward) auxin transport might also be reduced in osabcb14 shoots. Reduced acropetal PAT capacities and auxin levels in the mutant, however, are also in agreement with the described roles for AUX1 in acropetal auxin transport in Arabidopsis root (Yu and Wen, 2013). Therefore, this result also adds to the growing evidence supporting a major role of ABCBs in polar auxin transport.
In summary, our study provides convincing evidence for OsABCB14 as a plasma membrane auxin influx transporter (Figures 1, 3, 4 and 5); however, surprisingly we found that OsABCB14 is also involved in ion homeostasis. Fe, Mn, Cu, Zn, Mg and Ca are important nutrient elements for plant growth and development. The Fe concentrations in shoots, roots and seeds of osabcb14 mutants were significantly enhanced (Figure 6a), and the expression level of Fe deficiency-responsive genes was significantly upregulated in osabcb14 mutants (Figures 7a, S4a). Thus, increased Fe concentrations in shoots and roots of osabcb14 mutants (14 days old) are not only the result of higher Fe concentrations in seeds, but are also the result of the upregulated expression level of Fe deficiency-responsive genes. In addition, osabcb14 mutants displayed an insensitive phenotype and less reduced gene expression level to –Fe (Figures 6b–e, 7b and S4b), and increased Mn, Cu, Zn and Mg concentrations (Figure S3). Together, these results suggest that the disruption of OsABCB14 could negatively affect Fe concentrations and the homeostasis of other metal ions. Seeds of osabcb14 mutants have higher Fe concentrations (56% higher in osabcb14–1 and 43% higher in osabcb14–2; Figure 6a), indicating that osabcb14 mutants could be used for creating Fe-enriched crops. In the dicot Arabidosis auxin importer AUX1 was also shown to be involved in Fe deficiency, and the enhanced elongation of lateral roots in response to local Fe was demonstrated to depend on AUX1 action (Chen et al., 2010; Giehl et al., 2012). Thus, OsABCB14 may affect ion transport though the regulation of downstream ion transporters. A systematic transcriptome analysis of osabcb14 mutants grown under CK and –Fe will elucidate/rule out related genes in Fe and auxin signaling systems.
Plant materials and growth conditions
Rice plants (Oryza sativa L.) were grown in normal culture solution containing 1.425 mm NH4NO3, 0.323 mm NaH2PO4, 0.513 mm K2SO4, 0.998 mm CaCl2, 1.643 mm MgSO4, 0.25 mm NaSiO3, 0.009 mm MnCl2, 0.019 μm H3BO3, 0.152 μm ZnSO4, 0.155 μm CuSO4, 0.075 μm (NH4)6Mo7O24 and 0.125 mm EDTA-Fe(II) (Yoshida et al., 1976), without (CK) or with treatments (pH 5.2–5.5). Rice plants were grown in growth chambers with 60–70% humidity and a light/dark cycle of 12/12 h at 30/24°C. For transient expression, N. benthamiana plants were grown in vermiculite containing Murashige and Skoog salt nutritional liquid in a growth chamber with 60–70% humidity and a light/dark cycle of 12/12 h at 25/18°C.
Identification of the osabcb mutants
Two TOS17 insertion lines for OsABCB14, osabcb14–1 (NF6030) and osabcb14–2 (NG3129) were obtained from the Rice Genome Resource Center in Japan. The homozygous lines were screened by PCR, using primer TOS17-tail6 to confirm the integration of TOS17 in two mutant lines, and gene-specific primers ABCB14-1U/L and ABCB14-2U/L to identify wild-type (NIP)-bound OsABCB14. The PCR insertion products were ligated with pMD19–T Simple Vector (TaKaRa, http://www.takara-bio.com) and transformed into Escherichia coli DH5α, and the flanking sequences of the TOS17 insertion site were sequenced by Invitrogen (now Life Technologies, http://www.lifetechnologies.com). To confirm that the transcription level of the OsABCB14 gene in the NIP and two TOS17 homozygous lines, RT–PCR was performed using three pairs of primers, RTABCB14U1/L1, RTABCB14U2/L2 and RTABCB14U3/L3. qRT-PCR was performed using another three pairs of primers, qRTABCB14U1/L1, qRTABCB14U2/L2 and qRTABCB14U3/L3. Primer sequences for the PCR and RT-PCR are listed in Table S1.
The OsABCB14 (Os04g38570) coding region was amplified from full-length cDNA (AK103526) using primers ABCB14-OU/L (Table S1) for cloning into pH7FGW2 to create the ABCB14:EGFP fusion construct, following the manufacturer's instructions (Invitrogen). The final construct 35S:ABCB14-EGFP was transiently expressed in the leaves of N. benthamiana plants and onion, as previously described (Qi et al., 2012). After 2 days, the fluorescence was visualized with confocal microscopy (Leica TCS SP5; Zeiss, http://www.zeiss.com), as described by Guo et al. (2003). These constructs were also introduced into Agrobacterium tumefaciens strain EHA105 using electroporation and transferred into osabcb14–1 and osabcb14–2 using the callus infection method (Hiei et al., 1994) for complementation constructs. To analysis the transcription level of the OsABCB14 gene in the complementation lines, the primers RTABCB14U1/L1 and qRTABCB14U1/L1 listed in Table S1 were used.
To construct ProOsABCB14:GUS, a 2.1–kb promoter region of the OsABCB14 gene was amplified by PCR using primers ABCB14-proU/L (listed in Table S1) for cloning into the SalI–KpnI site of pBI101.3-GUS-plus. The final construct ProOsABCB14:GUS was introduced into A. tumefaciens strain EHA105 using electroporation and transferred into NIP using the callus infection method (Hiei et al., 1994).
Analysis of IAA concentrations and transport
Free IAA concentrations in seedling tissues were performed as described previously (Shen et al., 2013). Briefly, 20 mg of fresh shoots or roots of 7–day-old seedlings grown on normal nutrient solution were washed by sterile deionized water several times. The samples were then ground into fine powder in liquid nitrogen and dissolved in 50 mm KH2PO4–NaOH with 0.02% (w/v) ascorbic acid, then 250 pg of [13C]IAA was added to each sample solution. Free IAA concentrations were measured by gas chromatography-selected reaction monitoring mass spectrometry. The DR5:GUS construct was transformed into NIP and osabcb14 mutants to detect auxin distribution in the T2 generation. For staining of DR5:GUS seedlings, 100 mm sodium phosphate buffer (pH 7.0) containing 0.1% v/v Triton X–100 and 2 mm X–Gluc was used. Tissues were vacuum infiltrated for 15 min with staining solution and incubated for 30 min at 37°C before being observed using a Leica TCS SP5 microscope (Zeiss). Analyses of polar [3H]IAA transport were performed according to the method described by Qi et al. (2008).
Yeast auxin loading experiments were performed as described by Kamimoto et al. (2012). In brief, JK93da transformants were grown to OD600 = 1, washed and incubated at 30°C with 1 ml ml−1 5–[3H]IAA (specific activity 7.4 × 1011 Bq mmol−1; American Radiolabeled Chemicals, http://www.arc-inc.com) and [3H]BA (9.3 × 1011 Bq mmol−1; American Radiolabeled Chemicals) in SD media (pH 5.5). Aliquots of 1 ml were filtered twice with cold water after 0 and 10 min, respectively, and the retained radioactivity was quantified by scintillation counting. Rice protoplasts were prepared as described in Zhang et al. (2011), with the exception that leaf digests were performed overnight. Transport assays were performed as described in Henrichs et al. (2012). In brief, intact protoplasts were loaded by incubation with 1 μl ml−1 [3H]IAA (specific activity 7.4 × 1011 Bq mmol−1; American Radiolabeled Chemicals) and 4–[3H]NAA (9.3 × 1011 Bq mmol−1; American Radiolabeled Chemicals), in the presence of 100 nm IAA on ice. Import was started by incubation at 25°C, and was halted 10 min later by centrifugation with silicon oil. For export assays, loading was performed for 10 min on ice, allowing equal loading, and external radioactivity was removed by Percoll gradient centrifugation. Imported/exported radioactivity was determined by scintillation counting of the protoplast interfaces/supernatants, respectively, and is presented as the relative export/import of the initial export/uptake (export/import prior to temperature incubation).
Metal ion concentrations assay
Rice seedlings grown in hydroponics to 2 weeks of age and peeled rice seeds were analyzed to determine their metal ion concentrations with inductively coupled plasma mass spectrometry (Agilent 7500ce; Agilent Technologies, http://www.home.agilent.com), as previously described (Jia et al., 2011). Five biological replicates were performed for each sample.
Measurement of chlorophyll concentrations
The leaves of NIP and osabcb14 mutants grown in CK and –Fe for 2 weeks were collected and weighed for their fresh weight. Chlorophyll was extracted with ethanol for 24 h and the concentration was assayed based on the absorbance of the extract at 645, 652 and 663 nm (Wintermans and De Mots, 1965).
RT-PCR and qRT-PCR
The methods of total RNA extraction, reverse transcription and RT-PCR were as described in a previous report (Qi et al., 2012). Primer sequences for auxin-responsive genes and Fe deficiency-responsive genes are listed in Tables S2 and S3, respectively.
This work was supported by the National Science and Technology Support Plan (2012BAC09B01), the National Natural Science Foundation of China (31271692, 31171462 and 31371591), the Natural Science Foundation for Distinguished Young Scholars of ZheJiang province, China (LR13C130002), the State Key Laboratory of Molecular Developmental Biology, China, and funds of the Novartis Foundation and the Swiss National Funds (to M.G.). We gratefully acknowledge Professor Akio Miyao of the Rice Genome Resource Center (RGRC) in Japan for providing the full-length cDNA clones of OsABCB14, osabcb14–1 and osabcb14–2 mutants, and the technical assistance of Laurence Charrier.