Research

Mediator subunit 16 functions in the regulation of iron uptake expression in Arabidopsis

Yue Zhang*, Huilan Wu*, Ning Wang, Huajie Fan, Chunlin Chen, Yan Cui, Hongfei Liu and Hong-Qing Ling The State Key Laboratory of Plant Cell and Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing 100101, China

Summary Author for correspondence: Iron is an essential nutrient for plant growth and development, and its absorption is tightly Hong-Qing Ling controlled. Under iron limitation, FIT dimerizes with the four Ib bHLH proteins and activates Tel: +86 10 64806570 the expression of iron uptake . However, how the dimerized complex activates down- Email: [email protected] stream genes remains unclear. Received: 4 March 2014 Using forward genetics, a low-iron-sensitive mutant was screened. The corresponding gene Accepted: 22 April 2014 (MED16) was isolated, and its biological functions in iron homeostasis were characterized using approaches such as gene expression, protein subcellular localization, protein–protein New Phytologist (2014) 203: 770–783 interaction and chromatin immunoprecipitation assay. doi: 10.1111/nph.12860 Lesion of MED16 significantly reduced FRO2 and IRT1 expression in Arabidopsis roots. The MED16 mutants showed a low shoot iron concentration and severe leaf chlorosis under iron Key words: Arabidopsis, iron homeostasis, limitation, whereas it grew normally as wild-type under iron sufficiency. Furthermore, we low-iron-sensitive mutant, subunit showed that MED16 interacted with FIT and improved the binding of the FIT/Ib bHLH com- 16, regulation of iron uptake gene plex to FRO2 and IRT1 promoters under iron-deficient conditions. Additionally, we found that expression. many iron-deficient response genes, which are regulated by FIT, were also controlled by MED16. In conclusion, MED16 is involved in the iron deficiency response, and modulates the iron uptake gene expression under iron limitation. Our results increase the understanding of the molecular regulation mechanisms underlying iron uptake and homeostasis in plants.

responsible for this process and play a key role in iron uptake, Introduction because their lesion mutations cause seedling lethality under iron Iron is an essential micronutrient for plant growth and develop- deficiency (Eide et al., 1996; Robinson et al., 1999; Connolly ment and plays crucial roles in many fundamental biochemical et al., 2002; Henriques et al., 2002; Varotto et al., 2002; Vert processes in living cells, such as respiration and photosynthesis et al., 2002). The expression of FRO2 and IRT1 are known to be (Briat et al., 1995). Iron deficiency is one of the most common regulated by bHLH transcription factors (Colangelo & Guerinot, nutrient deficiencies in the world due to its low bioavailability in 2004; Jakoby et al., 2004; Yuan et al., 2005, 2008; Wang et al., alkaline soil, which accounts for approximately one third of the 2012). FIT (FER-like iron deficiency-induced transcription fac- world’s soils (Mori, 1999). Under the iron-deficient stress condi- tor), an ortholog of tomato FER in A. thaliana (Ling et al., 2002; tion, plants display chlorosis in young leaves and inhibition of Yuan et al., 2005), is a master regulator controlling FRO2 and plant growth and development. Additionally, excessive iron IRT1 expression, as well as the iron deficiency responses of strat- induces hydroxyl radical formation and damages cellular constit- egy I plants (Colangelo & Guerinot, 2004; Jakoby et al., 2004; uents, such as DNA and lipids (Halliwell & Gutteridge, 1992). Yuan et al., 2005, 2008). Loss-of-function FIT mutants fail to Therefore, the uptake, translocation and internal distribution of activate the iron deficiency response (such as FRO2 and IRT1 iron are tightly controlled in plants. expression) and show lethality under normal culture conditions. Under iron limitation, strategy I plants (Marschner, 1995) The Ib subgroup bHLH (Ib bHLH) proteins, which include activate the plasma-membrane ferric-chelate reductase(s) to bHLH38, bHLH39, bHLH100 and bHLH101, are required for reduce ferric iron to ferrous iron on the root surface, and the high activation of iron deficiency responses and iron uptake (Yuan affinity iron transporter takes up Fe2+ into the root cells. In the et al., 2008; Wang et al., 2012). Recently, Fan et al. (2014) model plant Arabidopsis thaliana, the genes FRO2 (Ferric showed that expression of the four bHLH genes was negatively Reductase/Oxidase 2) and IRT1 (Iron-Regulated Transporter 1) are regulated by SKB1, which is an epigenetic negative modulator associated with chromatin of the four Ib subgroup bHLH genes, *These authors contributed equally to this work. mediating their histone H4R3 dimethylation (H4R3sme2)

770 New Phytologist (2014) 203: 770–783 Ó 2014 The Authors www.newphytologist.com New Phytologist Ó 2014 New Phytologist Trust New Phytologist Research 771 according to the iron status of plants. The four Ib bHLH pro- conditions. The T-DNA insertion mutants (sfr6-2/med16-2 teins, possessing functional redundancy, dimerize with FIT and (SALK_048091) and sfr6-3/med16-3 (CS859103), Knight et al., activate FRO2 and IRT1 expression (Yuan et al., 2008; Wang 2009) were obtained from ABRC (Arabidopsis Biological et al., 2012). However, how the dimerized complex activates the Resource Center) and confirmed by PCR analysis. med16-3ox29/38 expression of its downstream genes remains unclear. was generated by crossing med16-3 with ox29/38 (a line overex- Mediator is a large protein complex comprising > 20 subunits, pressing FIT and bHLH38). med16-5ox29/38, which showed a which are organized into head, middle and tail modules (Asturias similar phenotype as med16-4 and in which the mutation was et al., 1999). Mediator functions as a bridge connecting the RNA confirmed by sequencing, was isolated by screening the EMS- polymerase II (Pol II) complex and specific transcriptional activa- mutagenized M2 population of ox29/38 on half-strength MS tors during the transcription process. Depending on the associ- agar plates without iron supply. ated protein components, Mediator serves as either a transcriptional activator or repressor (B€ackstr€om et al., 2007; Plant growth conditions Mathur et al., 2011; Zhang et al., 2012). In A. thaliana, 21 con- served and six A. thaliana-specific Mediator subunits have been Seeds of A. thaliana (ecotypes Col-0 (Columbia) and Ws), identified (B€ackstr€om et al., 2007). Among them, several Media- mutants, and transgenic and T-DNA insertion lines were surface- tor subunits have been implicated in specific signaling pathways, sterilized using 10% commercial bleach for 15 min and washed such as MED25/PFT1 in flowering time (Cerdan & Chory, three times with sterilized water. After vernalization at 4°C in the 2003; B€ackstr€om et al., 2007), MED14/SWP in cell proliferation dark for 3 d, seeds were suspended in 0.1% agar and sown on (Autran et al., 2002; B€ackstr€om et al., 2007), MED8 and half-strength MS (MS basal salt, Sigma, USA) agar plates (2% MED21 in JA-dependent defense responses (Dhawan et al., sucrose, 0.9% agar) buffered with 0.5 g l 1 4-Morpholineethane- 2009; Doares et al., 2009), and MED17, MED18 and MED20a sulfonic acid (MES) and adjusted to pH 5.8 with 1 M KOH. in small and long noncoding RNA production (Kim et al., 2011). Seedlings were grown at 23°C with a photoperiod of 16 h : 8 h, MED16, organized in the tail module of Mediator, is con- light : dark. Six days later, seedlings were used for further treat- served in yeast, animal and plants (B€ackstr€om et al., 2007). As a ments and analyses. For phenotypic analysis on soil, seeds of Ws specific binding partner of the DIF (differentiation-induced fac- and the low-iron-sensitive mutant obtained by genetic screening tor)-related transcriptional activator proteins, MED16 functions were sown directly on alkaline soil (+ 1.0% CaO) with or with- in the control of lipopolysaccharide-induced gene expression in out 100 lM Fe-EDTA watering and control soil, and were Drosophila melanogaster (Kim et al., 2004). In plants, MED16 of grown for 2 wk. Arabidopsis was first characterized as a positive activator of accli- For gene expression analysis, ferric-chelate reductase activity mation to freezing temperature after exposure to low temperature and Western blot analysis, 6-d-old seedlings were transferred to and was termed SFR6 (SENSITIVE TO FREEZING 6, Warren half-strength MS agar plates, which were buffered with MES with et al., 1996; Knight et al., 2009). SFR6 recruits CBF (C-box or without 50 lM Fe-EDTA, and grown for another 6 d. For binding factor) transcription factors into the nucleus and modu- phenotypic analysis and quantification of Fe, Zn and Mn con- lates cold on-regulated (COR) gene expression. Additionally, tents, the seedlings were transferred to iron-deficient and iron- Knight et al. (2008) showed that MED16/SFR6 is involved in sufficient half-strength MS agar plates and grown for 10 d. regulation of the circadian clock and controlling flowering time. Recently, Zhang et al. (2012) demonstrated that MED16/SFR6, Digital gene expression analysis as an essential positive regulator, functioned in salicylate-medi- ated systemic acquired resistance and jasmonate/ethylene- Six-day-old seedlings were transferred to half-strength MS agar induced defense pathways. plates with or without iron supply and grown for 6 d. Subse- In this study, we identified a low-iron-sensitive mutant quently, their roots were harvested in three independent repli- through genetic screening. Characterization of the mutant cates, and total RNA was isolated from each sample using revealed that the gene MED16 was mutated and the loss function TRIzol (Invitrogen, Carlsbad, CA, USA) according to the of MED16 resulted in sensitivity to iron limitation and a signifi- manufacturer’s instructions. The residual DNA in the RNA cant reduction of the iron uptake gene expression. Our results samples was removed by treatment with RNase-free DNase I suggest that MED16 plays an important role in the activation of (New England BioLabs, Ipswich, MA, USA) for 30 min at iron deficiency responses and the expression of iron uptake genes 37°C. Furthermore, poly(A) mRNA was isolated using oligo under iron limitation. (dT) beads. First-strand cDNA was synthesized using reverse transcriptase with random hexamer primers, and second-strand Materials and Methods cDNA with RNase H (Invitrogen) and DNA polymerase I (New England BioLabs). cDNA libraries were constructed and sequenced according to Illumina’s protocols (http://www.illu- Mutant lines mina.com/). DEGs were analyzed using edgeR (empirical The low-iron-sensitive mutant med16-4 was isolated by screening analysis of digital gene expression in R; Robinson & Smyth, the EMS-mutagenized M2 population of Arabidopsis thaliana 2007; Robinson et al., 2010) with a two-fold relative change (L.) Heynh, ecotype Wassilewskija (Ws), under iron-deficient threshold (P Value < 0.05, false discovery rate < 0.001).

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described by Connolly et al. (2002). Subsequently, membranes Measurement of metal concentration were washed in 1 9 TBST buffer four times (10 min per wash) Wild-type and mutants were treated on iron-deficient and iron- and then incubated for 1 h with goat anti-rabbit IgG conjugated sufficient media as described above for 10 d. The shoots were to horseradish peroxidase diluted 1 : 10 000. Chemiluminescence then harvested and incubated in 10 mM CaSO4EDTA for was performed using the Immobilon Western Chemiluminescent 10 min to eliminate the attached metal ions on the surface. After HRP Substrate (Millipore) according to the manufacturer’s washing three times with distilled water, samples were dried in an instructions and exposed to X-ray film (Kodak, Rochester, NY, oven at 80°C for 72 h. Subsequently, the Fe, Mn and Zn concen- USA). trations of each sample were determined using Inductively Cou- pled Plasma-Optic Emission Spectroscopy (ICP–OES; Perkin Bimolecular fluorescence complementation assay Elmer Inc., Waltham, MA, USA), as described by Yuan et al. (2008). The means and standard deviations were calculated from The coding sequences of MED16 were amplified using the three biological replications. primers described in Table S1 and fused to the 50 end of the N-ter- minal fragment of YFP (nYFP) to generate the construct MED16- nYFP, while the coding sequences of FIT, bHLH38, bHLH39, Quantitative real-time PCR bHLH100 and bHLH101 were amplified and separately ligated to After eliminating DNA contamination by DNase I (Thermo the C-terminal fragment of CFP (cCFP) to generate constructs Fisher Scientific, Waltham, MA, USA) at 37°C for 30 min, FIT-cCFP, bHLH38-cCFP, bHLH39-cCFP, bHLH100-cCFP mRNAs were converted to cDNAs using M-MLV reverse trans- and bHLH101-cCFP. Different plasmid combinations of criptase (Invitrogen) according to the manufacturer’s instruc- MED16-nYFP with FIT-cCFP, bHLH38-cCFP, bHLH39-cCFP, tions. Quantitative real-time PCR was performed using the bHLH100-cCFP or bHLH101-cCFP were transformed into leaf LightCycler (Roche Diagnostics). The gene-specific primers mesophyll protoplasts using PEG transformation (Sheen, 2001). (Supporting Information Table S1) and SYBR Green master mix The transformed protoplasts were incubated in the light at 22– (Takara, Dalian, Liaoning, China) were used according to the 25°C for 18–20 h and then screened using a confocal microscope manufacturer’s protocol in a final volume of 10 ll. The relative (LSM710NLD; Carl Zeiss, Oberkochen, Germany), as described expression level of each candidate gene was calculated using the previously (Yuan et al., 2008). DD 2 CT method with ACTIN8 as the internal reference gene. Generation of the yeast sin4/med16 mutant and transcrip- Assay of root ferric-chelate reductase activity tional activation assay in yeast cells The ferric-chelate reductase activity of the whole intact root sys- The yeast (Saccharomyces cerevisiae) strain INVSc1 (His , Leu , tem was determined as described by Kong et al. (2013). Roots of Trp , Ura ; Invitrogen) was used to generate the MED16 seedlings grown on half-strength medium with or without iron mutant. SIN4 is a homolog of Arabidopsis MED16 in the yeast supply for 6 d were harvested and rinsed with distilled pure water. genome and can easily be deleted by homologous recombination The roots were then transferred to assay solution (0.2 mM using a PCR-based gene deletion approach (Reid et al., 2002). CaSO4 2H2O, 0.1 mM FeCl3, 0.1 mM HEDTA, 0.2 mM The deletion strain was generated using a chimeric primer PCR BPDS, 5 mM MES, pH 5.5) at room temperature in the dark- technique, in which the URA selectable marker is amplified by ness. After 30 min, an aliquot of the assay solution was removed, PCR and attached to a 40-bp homologous region from the 50 and the absorbance was determined using a spectrometer at and 30 side of the coding region of SIN4. The primer used to 535 nm. The Fe(II)-BPDS concentration was calculated using construct the gene deletion is listed in Table S1. Gene disruption the extinction coefficient of 22.14 mM 1 cm 1. The experiment was achieved by transformation of the PCR products into yeast was repeated three times independently. cells using the LiOAc transformation procedure (Invitrogen). Selection of the sin4 mutant was performed on SC-U medium, and deletion of SIN4/MED16 was confirmed by RT-PCR. Western blot analysis For transcriptional activation assay, the plasmids pAD- Western blot analysis of IRT1 was performed as described previ- bHLH38, pBD-FIT-pFRO2:GUS and pBD-pFRO2:GUS, con- ously (Yuan et al., 2008). Briefly, total proteins were extracted structed by Yuan et al. (2008), were used in this experiment. Pairs from roots grown on Fe-deficient and iron-sufficient media, as of pBD-FIT-pFRO2:GUS or pBD-pFRO2:GUS with pAD- already described. The protein samples were separated by SDS– GAL4 or pAD-bHLH38 were separately transformed into wild- PAGE and transferred to a PVDF membrane (GE Healthcare, type and sin4 mutant yeast cells. GUS staining was performed as Pittsburgh, PA, USA) using semi-dry transfer (Bio-Rad). The described by Yuan et al. (2008). membranes were blocked in 1 9 TBST buffer with 5% nonfat dry milk for 2 h at room temperature (or overnight at 4°C) and Chromatin immunoprecipitation (ChIP) assay then hybridized for 1 h with 1 : 15 000-diluted IRT1 peptide antibody, which was raised against the chimeric fusion protein of For quantitative ChIP-PCR assays, the roots of 35S:FIT-GFP the GST tag and the peptide PANDVTLPIKEDDSSN, as (expressing the fusion protein FIT-GFP in wild-type Col-0),

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35S:MED16-GFP (expressing the fusion protein MED16-GFP Results in wild-type Col-0) and 35S:FIT-GFP/med16-3 (expressing the fusion protein FIT-GFP in med16-3) were harvested after grow- Isolation of low-iron-sensitive mutant in Arabidopsis ing on half-strength MS agar plates without iron supply for 7 d. ChIP was performed as described by Bowler et al. (2004). The In order to identify iron homeostasis mutants, seeds of A. thaliana antibody against GFP was used to immunoprecipitate DNA/pro- ecotype Ws were mutagenized with ethyl methanesulfonate tein complexes from the chromatin preparation. DNA precipi- (EMS), and the M2 population was screened on half-strength MS tated from the complexes was recovered, purified and analyzed agar medium without iron. We identified a mutant which showed using the multiple-quantitative ChIP-PCR method, as described more severe chlorosis than wild-type under iron limitation and by Fan et al. (2014). The primers were designed to amplify frag- finally died due to iron deficiency, whereas it grew similar to wild- ments of 140–390 bp within the chromatin region of genes and type plants in the presence of iron (Fig. 1a). Additionally, the are provided in Table S1. mutant showed paler and larger cotyledons and a longer

(a) Ws Mutant (c) Ws Mutant

Control

+Fe

1.0% CaO

–Fe Fig. 1 Phenotypic characterization of the 1.0% CaO + 100 µM FeEDTA low-iron-sensitive mutant of Arabidopsis. (a) Six-day-old seedlings of wild-type (Ws) and (b) Col fit1-2 irt1 Mutant Ws Col sfr6-2 sfr6-3 Mutant Ws its low-iron-sensitive mutant (mutant) grown (d) on half-strength MS agar plate with (+Fe) and without iron supply (Fe) for 10 d. (b) Phenotypic comparison among the low-iron- sensitive mutant, fit1-2, irt1 and their wild- types Col and Ws. Six-day-old seedlings of Col, fit1-2, irt1, the low-iron-sensitive mutant (mutant) and Ws grown on half- strength MS agar plate with (+Fe) and without iron supply (Fe) for 10 d. (c) Phenotypic characterization of the low-iron +Fe sensitive mutant on alkaline soil. The mutant +Fe and its wild-type Ws were directly sown, germinated and grown on alkaline soil (normal garden soil + 1.0% CaO) with (1.0% CaO + 100 lM Fe-EDTA) or without 100 lM Fe-EDTA watering (1.0% CaO) and on normal garden soil (control) for 2 wk. (d) Phenotype comparison among the low-iron- sensitive mutant (mutant), sfr6-2 and sfr6-3 and their wild-types Ws and Col. Six-day-old seedlings grew on half-strength MS agar plate with (+Fe) and without iron supply –Fe –Fe (Fe) for 10 d.

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hypocotyledonary axis than did wild-type under normal growth (a) conditions (Fig. S1a) and a light-green leaf color when grown on soil (Fig. S1b). Subsequently, we compared the phenotype and sensitivity to iron deficiency of the mutant with those of the classic iron homeostasis mutant fit1-2 (Yuan et al., 2005) and irt1 Col sfr6-3 (SALK_054554C). As shown in Fig. 1(b), the mutant showed similar chlorotic phenotypes and sensitivities as fit1-2 and irt1 under iron deficiency. Furthermore, we examined its growth on alkaline soil (garden soil supplemented with 1.0% CaO (w/w)). Consistent with the phenotype observed on iron-deficient mutant x sfr6-3 medium, the mutant displayed more severe leaf chlorosis and retarded growth than did wild-type, whereas no obvious growth difference was observed between the mutant and wild-type under normal growth conditions (garden soil without supplementation of CaO) (Fig. 1c, upper and middle sections). Additionally, the Ws mutant chlorotic phenotype of the mutant on alkaline soil was rescued by providing iron solution to the soil (Fig. 1c, bottom part). These results indicated that the mutant is a low-iron-sensitive mutant and that its iron homeostasis was affected under iron-deficient conditions. (b) Ws mutant 35S:MED16-GFP For genetic analysis, we crossed the mutant with its wild-type. The F2 plants revealed a 3 : 1 segregation ratio of wild-type over the mutant, indicating that the low-iron-sensitive mutant harbors a monogenetic recessive mutation in a nuclear gene.

The gene responsible for the low-iron-sensitive mutant encodes MED16 In order to clone the gene responsible for the low iron sensitivity, the mutant was crossed with ecotype Columbia (Col-0). In the F2 population, plants with the low-iron-sensitive phenotype were selected and used for genetic mapping. After analysis of 2869 F2 mutant plants, the gene responsible for the low iron sensitivity Fig. 2 Allelic analysis of Arabidopsis mutants and phenotypic was mapped between markers SSLP1475 and NGA8 in the cen- characterization of the complementation line. (a) Allelic analysis between the low-iron-sensitive mutant and sfr6-3. Phenotypes of Col, Ws, sfr6-3, tromeric region of chromosome 4 (Fig. S2a). The physical dis- the low-iron-sensitive mutant (mutant), and F1 progenies (mutant 9 sfr6- tance between the two markers was 3.1 Mbp. The recombination 3) derived from the cross of sfr6-3 with the low-iron-sensitive mutant frequency, consistent with previous reports (Knight et al., 2009; under iron deficiency. The representative photo was taken at 10 d after Zhang et al., 2012), was very low in this region, which compli- growth on half-strength MS agar medium without iron supply. (b) cated further fine mapping of the gene . Thus, we compared Phenotypic characterization of the complementation line under iron limitation. Phenotypes of wild-type (Ws), the low-iron-sensitive mutant the phenotype of our low-iron-sensitive mutant with those of the (mutant) and its complementation line (35S:MED16-GFP) grown on half- mutants described in this region. Mutants sfr6-1, sfr6-2, sfr6-3 strength MS agar medium without iron supply for 10 d. (Knight et al., 2009) and med16-1/ien1 (Zhang et al., 2012) of the Mediator subunit 16 gene (MED16,alsoknownasSENSITIVE result, we generated the 35S:MED16-GFP construct and trans- TO FREEZING 6 (SFR6)), similar to our mutant, showed paler formed it into the mutant. Transgenic plants, similar to wild-type, cotyledons under normal culture conditions, suggesting that our grew normally on iron-deficient MS agar plates (Fig. 2b), indicat- mutant is allelic to sfr6 and med16-1/ien1. To confirm this ing that the defective functions in the low-iron-sensitive mutant hypothesis, we obtained the SALK_048091 (sfr6-2)and were complemented by expressing wild-type MED16 cDNA. CS859103 (sfr6-3) lines, which carry a T-DNA insertion in These results further demonstrated that MED16 is the target gene, At4 g04920 (MED16/SFR6,Knightet al., 2009), from the Ara- and its mutation is responsible for the low-iron-sensitive pheno- bidopsis Biological Resource Center (ABRC) and compared their type. To avoid terminology confusions in further analyses, we phenotypes. Similar to our mutant, sfr6-2 and sfr6-3 displayed renamed the mutant sfr6-2 as med16-2, sfr6-3 as med16-3, and severe leaf chlorosis under iron deficiency (Fig. 1d). Subsequently, named our low-iron-sensitive mutant as med16-4 hereafter, based we crossed sfr6-3 with our mutant, and the F1 plants displayed the on Zhang et al. (2012), who called their ien1 mutant med16-1. same severe chlorosis as their parents under iron limitation In order to identify mutation(s), we amplified and sequenced (Fig. 2a). These results indicated that our low-iron-sensitive genomic MED16 DNA from med16-4 and its wild-type. A single mutant is an allelic mutant of sfr6-3. To further confirm this base transition from G to A was detected at the junction between

New Phytologist (2014) 203: 770–783 Ó 2014 The Authors www.newphytologist.com New Phytologist Ó 2014 New Phytologist Trust New Phytologist Research 775 the seventh intron and the eighth exon by comparing DNA cultivation for 10 d, shoots were harvested and their iron concen- sequences between the mutant and wild-type (Fig. S2b), resulting tration was determined using ICP-OES. As shown in Fig. 3(a), in altered splicing of MED16 mRNA and decreased transcript the iron concentrations of med16-2, med16-3 and med16-4 were abundance (Fig. S2c). significantly lower (c. 50%) than that of wild-type under iron For physiological characterization, we determined the shoot deficiency, whereas no marked difference in the iron concentra- iron concentration of the mutants and their wild-types. Six- tion was observed under iron sufficiency. Furthermore, we mea- day-old seedlings of med16-2, med16-3 and its wild-type Col-0, sured Zn and Mn concentrations between the mutants and their and med16-4 and its wild-type Ws were transferred to half- wild-types, and no obvious difference was observed under iron- strength MS agar plates with 0 or 50 lM Fe-EDTA. After deficient or iron-sufficient conditions (Fig. 3b,c). These results support the hypotheses that MED16 functions in iron homeosta- (a) 160 sis, and that the severe chlorotic phenotypes of med16-2, med16- Col 3 and med16-4 under iron limitation are due to a lack of iron 140 med16-2 med16-3 in vivo. 120 Ws med16-4 100 Expression profiles of MED16 and subcellular localization of 80 its product

60 In order to investigate the expression profiles of MED16, we per- formed quantitative reverse transcription-polymerase chain 40 Fe concentration (ppm) ** ** ** 20 3.5 (a) 0 +Fe –Fe 3 2.5 (b) 300 Col 2 250 med16-2 1.5 200 med16-3 Ws 1 150 med16-4 relative expression level 0.5 100

MED16 0 50 Root Leaf Flower Seed Zn concentration (ppm)

0 1.6 (b) +Fe –Fe 1.4 (c) 250 Col med16-2 1.2 med16-3 200 1.0 Ws med16-4 0.8 150 0.6

100 relative expression level 0.4

50 0.2 MED16 Mn concentration (ppm) 0.0 0 Leaf Root +Fe –Fe Fig. 4 Expression pattern of MED16 in Arabidopsis. (a) Quantitative RT- Fig. 3 Metal concentration determination in shoots of Arabidopsis mutants PCR analysis of MED16 expression in the indicated tissues of Arabidopsis. and their wild types. Iron (a), zinc (b) and manganese (c) concentrations in (b) The relative expression level of MED16 in leaves and roots under iron- shoots of Arabidopsis mutants (med16-2, med16-3, med16-4) and their sufficient and iron-deficient conditions. Six-day-old seedlings of Col grown wild-type counterparts (Col and Ws), which were cultivated on half- on half-strength MS agar plates with (+Fe; dark gray bars) or without iron strength MS agar plates with (+Fe) or without iron supply (Fe) for 10 d. supply (Fe; light gray bars) for 6 d. Total RNA was extracted, and the Values shown in the figure represent the means SD of three biological relative expression intensity of MED16 was determined by qRT-PCR. The y replicates. Significant differences compared with wild-type (measured by axis shows RNA levels normalized to that of ACTIN8. n = 3, values Student’s t-test): **, P < 0.01. represent means SD.

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reaction (RT-PCR) analysis. MED16 mRNA was detected in var- wild-type under iron-deficient conditions (Figs 1, 3a), indicating ious tissues, including root, leaf, flower and seed (Fig. 4a). that iron uptake in the mutants may be impaired. Therefore, we MED16 was ubiquitously expressed, and a high mRNA level was examined expression of FRO2 and IRT1 (two major iron uptake observed in seed. Given the Fe-related phenotype in med16,we genes in Arabidopsis) and FIT (a key regulator controlling FRO2 analyzed the response of MED16 expression to Fe status. As and IRT1 expression) using quantitative RT-PCR in wild-type shown in Fig. 4(b), a slight increase in MED16 expression was and med16 mutants. The expression of IRT1 and FRO2 observed in shoots and roots under iron deficiency compared to decreased significantly in the roots of med16 mutants under iron iron sufficiency, but the difference was not significant. Further- deficiency compared with their wild-type counterparts (Fig. 5a,b). more, we used the stable transgenic line expressing MED16-GFP Significantly increased expression of FIT was observed in med16 driven by the 35S promoter and investigated the subcellular mutants under iron sufficiency, whereas no obvious difference localization of the MED16 protein. Consistent with previous was detected between the mutants and wild-type under iron defi- reports (Knight et al., 2009), the fusion protein MED16-GFP ciency (Fig. 5c). We also analyzed the ferric-chelate reductase was localized in the nucleus in root tip cells (Fig. S3a). Addition- (FCR) activity and IRT1 protein accumulation of med16 ally, we found that the MED16-GFP signal was predominantly mutants and their wild-type counterparts. As shown in Fig. 5(d), in the nuclei of stomata cells of leaves (Fig. S3b). FCR activity of med16-2, med16-3 and med16-4 was significantly lower than wild-types under iron deficiency, while no difference was observed between the mutants and wild-types under iron suf- MED16 is involved in the regulation of IRT1 and FRO2 ficiency. As shown in Fig. 5(e,f), IRT1 protein accumulation was expression detected in roots of both the med16 mutants and wild-types As shown above, med16 mutants showed sensitivity to iron limi- under iron deficiency, but the IRT1 level in med16 mutants was tation and a low shoot iron concentration compared with the significantly lower than wild-type. These results suggest that

(a)3.5 (b) 1.4 Col Col 3.0 med16-2 1.2 med16-2 med16-3 med16-3 1.0 2.5 Ws Ws 2.0 med16-4 0.8 med16-4

1.5 0.6

1.0 0.4 relative expression level relative expression level **** ** 0.5 ** 0.2 ** ** IRT1 FRO2 0.0 0.0 +Fe –Fe +Fe –Fe

(c)0.4 (d) Col med16-2 med16-3 3.5 Fig. 5 Expression profiles of IRT1, FRO2 and Ws med16-4 Col FIT in roots of med16 mutants of Arabidopsis 0.3 3.0 med16-2 and their wild-type counterparts. The relative med16-3 expression abundance of (a) IRT1, (b) FRO2 0.3 Ws ** –1 2.5 and (c) FIT in roots of med16 mutants h med16-4 (med16-2, med16-3, med16-4) and wild- 0.2 –1 ** 2.0 types (Col and Ws) under iron-sufficient ** ** ** (+Fe) and iron-deficient (Fe) conditions 0.2 1.5 ** using qRT-PCR analysis. The y-axis shows

0.1 mol Fe(II) g RNA levels normalized to that of ACTIN8.

μ 1.0 relative expression level n = 3, values represent means SD. (d)

FIT 0.1 0.5 Determination of ferric-chelate reductase

Ferric-chelate reductase activity activity in roots of med16 mutants and their 0.0 0.0 wild-types under iron-sufficient (+Fe) and +Fe –Fe +Fe –Fe iron-deficient (Fe) conditions. (e) Western blot analysis of IRT1 accumulation in roots of (e) (f) 30 000 mutants and their wild-types grown on half- 25 000 strength MS agar media with (+) or without l s Co med16-2med16-3W med16-4 20 000 (–) iron supply for 6 d. (f) The relative +– +– +– + – +– 15 000 intensity of IRT1 compared with the loading 10 000 control was obtained using IMAGE J. Values IRT1 5000 represent the mean from three independent Signal intensity 0 replicates. Significant differences compared +–+–+–+–+– Coomassie with wild-type (measured by the Student’s Col med med Ws med t-test): 16-2 16-3 16-4 **, P < 0.01.

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MED16 is involved in the regulation of FRO2 and IRT1 expres- FIT/Ib bHLH heterodimer. In this assay system, FIT, bHLH38, sion and iron uptake in Arabidopsis under iron limitation. and FIT together with bHLH38 of Arabidopsis were separately introduced and expressed in the yeast cells, and the FIT/ bHLH38 heterodimer, but not FIT/FIT or bHLH38/bHLH38, MED16 interacts with FIT and regulates the expression of activated transcription of GUS driven by the FRO2 promoter iron uptake genes (Yuan et al., 2008). To accomplish this, we knocked-out the FIT, a master transcription factor, regulates the expression of MED16-homolog gene SIN4 from the yeast genome (see Experi- FRO2 and IRT1 by forming a heterodimer with bHLH38, mental Procedures, Fig. S4) and analyzed GUS expression in sin4 bHLH39, bHLH100 and bHLH101 (Yuan et al., 2008; Wang yeast cells. Filter lift assays for GUS staining revealed strong blue et al., 2012). The null FIT-mutant fit1-2, similar to med16 color in wild-type yeast cells transformed with a plasmid combi- mutants (Fig. 5), showed a significant decrease in FRO2 and nation of pAD-bHLH38 and pBD-FIT-PFRO2:GUS, whereas the IRT1 expression at the mRNA and protein levels in roots under blue color was much weaker in sin4 mutant cells containing the iron deficiency (Yuan et al., 2005). Considering that MED16 is a same plasmid combination (Fig. 7a). Quantitative analysis con- subunit of the large complex Mediator which serves as a bridge firmed that knocking out SIN4 significantly reduced GUS expres- between the RNA polymerase II and the transcription factor in sion compared with wild-type (Fig. 7b). This result demonstrates the transcriptional machinery (Kornberg, 2005), MED16 may that the MED16-homolog SIN4 in yeast, similar to MED16 in link the FIT/Ib bHLH heterodimer with the Mediator complex Arabidopsis, is required for the expression of GUS driven by the to activate or enhance expression of downstream genes, such as FRO2 promoter PFRO2 under control of the FIT/bHLH38 com- FRO2 and IRT1. To test this hypothesis, we performed bimolec- plex in yeast cells. ular fluorescence complementation (BiFC) assays in Arabidopsis protoplasts. As shown in Fig. 6, MED16 interacted with FIT, MED16 functions in the recruitment of the FIT/Ib bHLH but not with bHLH38, bHLH39, bHLH100 or bHLH101, and complex to FRO2 and IRT1 promoters the interaction occurred specifically in the nucleus. We next used an artificial yeast transcriptional activation assay MED16, as shown above, is involved in the regulation of FRO2 system created by Yuan et al. (2008) to confirm that MED16 is and IRT1 expression through FIT and its counterpart bHLH38, required for activation of the iron uptake gene expression by the bHLH39, bHLH100 or bHLH101. To explore how MED16

GFP ChlorophoryII Bright Field Merged

MED16-nYFP + FIT-cCFP

MED16-nYFP + bHLH38-cCFP

MED16-nYFP + bHLH39-cCFP Fig. 6 Bimolecular fluorescence complementation assay of MED16 with FIT in Arabidopsis protoplasts. The Arabidopsis mesophyll protoplasts were transformed with MED16-nYFP + different plasmid combinations (MED16- bHLH100-cCFP nYFP with FIT-cCFP, bHLH38-cCFP, bHLH39-cCFP, bHLH100-cCFP or bHLH101-cCFP) and investigated under a confocal microscope after incubation for 16– 20 h. The protoplasts transformed with MED16-nYFP and FIT-cCFP displayed green MED16-nYFP + fluorescent signal in the nucleus, whereas no bHLH101-cCFP fluorescence signal was observed in the protoplasts transformed with other combinations.

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(a) Wild type sin4/med16 between FIT and bHLH38 (the same as wild-type) was detect- 1 2 able, indicating that formation of the FIT/Ib bHLH complex in pAD-GAL4/pBD-GAL4-P ::GUS FRO2 vivo is independent of MED16.

3 4 Considering that Mediator physically associates with both pAD-GAL4/pBD-FIT-P ::GUS FRO2 DNA-bound transcription factors and Pol II, and serves as an 5 6 integrative hub for transcriptional regulation (Kornberg, 2005; pAD-AtbHLH38/pBD-FIT-P ::GUS FRO2 Malik & Roeder, 2005, 2010; Conaway & Conaway, 2011; Lari- viere et al., 2012), we hypothesized that MED16 functions in the binding of the FIT/Ib bHLH complex to the promoters of target (b) 10 genes. To explore this hypothesis, a chromatin immunoprecipita- 9 8 tion (ChIP) assay was performed. Six-day-old seedlings of 35S: protein) 7 FIT-GFP (overexpressing the FIT-GFP fusion protein in wild- –1 6 type Col-0), 35S:MED16-GFP (overexpressing the MED16- mg ** –1 5 GFP fusion protein in wild-type Col-0) and 35S:FIT-GFP/ 4 med16-3 (overexpressing FIT-GFP fusion protein in the mutant

GUS content 3 2 med16-3) were transferred onto half-strength MS agar medium 1 without an iron supply and grown for 7 d. Chromatin was then (pmol MU min 0 extracted from the roots and immunoprecipitated using anti- 123456 GFP antibody, and the amount of FRO2 or IRT1 promoter Fig. 7 Effect of MED16 on GUS expression regulated by FIT/bHLH38 immunoprecipitated by anti-GFP antibody was determined using complex in yeast. Qualitative (a) and quantitative (b) assays of quantitative ChIP-PCR with primers specific for the FRO2 and transcriptional activation of GUS expression driven by the FRO2 promoter IRT1 promoter regions (Table S1). As shown in Fig. 8, precipi- via the FIT/bHLH38 complex in wild-type and sin4/med16 (mutant) yeast cells. Yeast cells transformed with the plasmid combinations pAD-GAL4/ tated DNA from the FRO2 and IRT1 promoters was significantly

pBD-GAL4-PFRO2::GUS in wild-type (1) and mutant (2), pAD-GAL4/pBD- lower in 35S:FIT-GFP/med16-3 than in 35S:FIT-GFP and 35S: FIT-PFRO2::GUS in wild-type (3) and mutant (4), and pAD-AtbHLH38/ MED16-GFP, whereas no difference was observed between 35S: pBD-FIT-PFRO2::GUS in wild-type (5) and mutant (6). The values in (b) FIT-GFP and 35S:MED16-GFP. These results clearly demon- represent the mean SD of three independent biological replicates. strated that MED16 functions in the recruitment of the FIT/Ib Significant differences compared with wild-type (No. 5) (measured by Student’s t-test): **, P < 0.01. bHLH complex to the FRO2 and IRT1 promoters, and loss of MED16 reduces the recruitment to FRO2 and IRT1 promoters, consequently decreasing FRO2 and IRT1 expression. functions in the transcriptional regulation process, we examined Yuan et al. (2008) reported that double overexpression of FIT the interaction of FIT with bHLH38 in mesophyll protoplasts of and bHLH38 in Arabidopsis resulted in the constitutive expres- the med16-3 mutant. As shown in Fig. S5, the interaction sion of IRT1 and FRO2, and the transgenic line ox29/38

0.7

0.6

0.5

0.4

0.3 ** ChIPsignal (% input) 0.2 **

** ** 0.1 ** ** ** ** ** ** *

0 FRO2-A FRO2-B FRO2-C FRO2-D FRO2-E FRO2-F IRT1-A IRT1-B IRT1-C IRT1-D IRT1-E ACTIN8 Fig. 8 ChIP analysis of FIT and MED16 recruitment to FRO2 and IRT1 promoters of Arabidopsis. Chromatin was extracted from 35S:FIT-GFP (white bars), 35S:MED16-GFP (light gray bars) and 35S:FIT-GFP/med16-3 (dark gray bars) seedlings after administration of low iron stress for 7 d, and then precipitated using anti-GFP antibody. Precipitated DNA was amplified with primers corresponding to the different sequence regions of the FRO2 and IRT1 promoters. The ChIP signal obtained from multiple-quantitative ChIP-PCR was quantified as the percentage of total input DNA. Three biological replicates were performed. Standard deviations were calculated from three technical repeats. FRO2-A, FRO2-B, FRO2-C, FRO2-D, FRO2-E, FRO2-F and IRT1-A, IRT1-B, IRT1-C, IRT1-D and IRT1-E indicate the amplified fragments from the promoter region of FRO2 and IRT1, respectively. ACTIN8 was used as a negative control. Significant differences compared with wild-type (measured by Student’s t-test): *, P < 0.05; **, P < 0.01.

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8 in shoots of med16-3ox29/38 and med16-5ox29/38 was also markedly

ox29/3 decreased compared with ox29/38 under iron-limited stress ox29/38 (Fig. 10a). Further analysis showed that the expression of IRT1 and FRO2 significantly decreased at the transcriptional level in ox29/38 ox29/38 md16-5 med16-3 med16-3 ox29/38 Col med16-3 and med16-5 compared with ox29/38 under normal and iron-deficient conditions (Fig. 10b,c). A significant reduction in FCR activity and IRT1 protein accumulation was also detected in med16-3ox29/38 and med16-5ox29/38 compared with ox29/38 under iron-sufficient and iron-deficient conditions (Fig. 10d–f). These results support the hypothesis that MED16 is involved in regulation of the constitutive expression of FRO2 and IRT1 in ox29/38. In order to identify potential candidate genes regulated by MED16, we performed Digital Gene Expression (DEG) analysis of med16-3 and its wild-type Col-0 under iron deficiency using RNA-seq and compared their transcriptomes. As shown in Tables S2 and S3, a number of genes regulated by iron deficiency were +Fe downregulated in root and shoot of the med16-3 mutant com- pared with wild-type. Interestingly, besides IRT1 and FRO2, the transcriptional levels of a set of FIT-dependent or partly depen- dent genes (Colangelo & Guerinot, 2004), such as MYB10, MYB72 and COPT2, were significantly downregulated in the roots of med16-3 (Table 1). These data suggest that transcription of these genes may be modulated by MED16.

Discussion In this study, we demonstrate that MED16, as a component of transcription machinery, is involved in the regulation of iron uptake gene expression through interaction with FIT in addition to its functions reported previously (Knight et al., 2008, 2009; –Fe Zhang et al., 2012). Loss-of-function of MED16 significantly Fig. 9 Phenotypic characterization of med16 mutants of Arabidopsis in the reduced expression of the major iron uptake genes IRT1 and ox29/38-genetic background. Phenotypic comparisons of wild-type (Col), FRO2 in root, and shoot iron concentration under iron defi- med16-3 (a MED16 mutant of Col), ox29/38 (overexpressing FIT and ciency. The mutants were more sensitive to iron limitation than ox29/38 AtbHLH38), med16-3 (a MED16 mutant line generated by a cross were wild-types (Fig. 1). between med16-3 and ox29/38) and med16-5ox29/38 (a MED16 mutant line screened from an EMS mutant population of ox29/38) grown with Previous studies showed that the expression of IRT1 and (+Fe) or without iron supply (Fe) for 10 d. FRO2 is controlled by the transcription factor FIT and the four Ib subgroup bHLHs (bHLH38, bHLH39, bHLH100 and bHLH101) (Colangelo & Guerinot, 2004; Yuan et al., 2005, accumulated more iron in the shoots and exhibited tolerance to 2008; Wang et al., 2012). Of them, FIT, as a master transcrip- iron deficiency. To explore whether constitutive expression of tion factor, interacts with bHLH38, bHLH39, bHLH100 or IRT1 and FRO2 in ox29/38 also requires MED16, we crossed bHLH101 to form heterodimer(s) and regulate gene expression med16-3 with ox29/38 and generated the MED16 mutant of iron-deficient responses and uptake, such as FRO2 and IRT1. med16-3ox29/38 in the genetic background of ox29/38 (Fig. S6a). Here, we demonstrate that MED16 is directly involved in the Furthermore, we screened the EMS-mutagenized M2 population regulation of IRT1 and FRO2 expression. First, med16 mutants, of the ox29/38 line, identified a novel MED16 mutant in the similar to the FIT mutant fit1-2, showed a chlorotic phenotype ox29/38-genetic background, and named it med16-5ox29/38.In (Fig. 1), low expression of FRO2 and IRT1 (Fig. 5) and low iron this mutant, a base transition from G to A in the 15th exon of concentration in shoots (Fig. 3a) under iron-limited stress. MED16 was observed, generating a premature stop codon (Fig. Expression profile analysis revealed that a mutation in MED16 S6b,c). Similar to med16-3, the med16-3ox29/38and med16-5ox29/38 did not affect FIT expression, and even an increased expression plants showed more sensitivity to iron deficiency with severe was observed under iron sufficiency (Fig. 5c). Second, BiFC chlorosis, and growth impairment on half-strength MS agar plate assays showed that MED16 interacted with FIT in vivo (Fig. 6). without iron supply, while no leaf chlorosis or stunted growth Third, quantitative ChIP-PCR analysis revealed that loss of was observed in ox29/38 plants under the same conditions MED16 function significantly reduced recruitment of FIT to (Fig. 9). Consistent with this phenotype, the iron concentration FRO2 and IRT1 promoters (Fig. 8). Additionally, the promoters

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(a) (b) 1000 5 Col Col med16-3 med16-3 ox29/38 ox29/38 800 med16-3ox29/38 med16-3ox29/38 4 med16-5ox29/38 med16-5ox29/38

600 3 ** ** 400 2 **

relative expression level relative ** Fe concentration (ppm) 200 1 ** ** IRT1 ** ** ** ** 0 0 +Fe –Fe +Fe –Fe

(c)2.5 (d) Col 4 med16-3 Col ox29/38 med16-3 2.0 ox29/38 med16-3ox29/38 med16-3ox29/38 ox29/38 3 med16-5 –1 ** med16-5ox29/38 1.5 h –1 **

2 ** 1.0 ** ** µmol Fe(II) µmol Fe(II) g relative expression level relative 1 0.5 ** ** ** FRO2

** ** Ferric-chelate reductase activity 0.0 0 +Fe –Fe +Fe –Fe

(e) (f) 25 000

20 000 ox29/38 ox29/38 15 000 Col med16-3 ox29/38 med16-3 med16-5 10 000 +–+–+–+–+– IRT1

Signal intensity 5000

Coomassie 0 + –+–+–+–+–

Fig. 10 Iron concentration and expression profiles of FRO2 and IRT1 in med16 mutants of Arabidopsis in the ox29/38-genetic background. (a) Shoot iron concentration of med16-3, med16-3ox29/38, med16-5ox29/38 mutants and their wild-type counterparts (Col, ox29/38) under iron sufficiency (+Fe) and iron deficiency (Fe). (b, c) Relative expression levels of (b) IRT1 and (c) FRO2 in roots of mutants and their wild-types grown on half-strength MS agar medium with (+Fe) or without iron supply (Fe) for 6 d. The y axis in (b) and (c) shows RNA levels normalized to that of ACTIN8 (n = 3). (d) Ferric-chelate reductase activity in roots of mutants and their wild-type counterparts, which were cultivated on half-strength MS agar medium with (+Fe) or without (Fe) iron supply for 6 d. (e) Western blot analysis of IRT1 accumulation in roots of mutants and their wild-type counterparts grown on half-strength MS agar media with (+) or without () iron supply for 6 d. (f) The relative intensity of IRT1 compared with the loading control was obtained using IMAGE J. Values represent the mean SD from three independent replicates. Significant differences compared with wild-type (measured by Student’s t-test): **, P < 0.01.

of FRO2 and IRT1 were immunoprecipitated by anti-GFP anti- directly, but rather to act as a co-activator of FIT and in the regu- body from chromatin extracted from roots of 35S:MED16-GFP, lation of FRO2 and IRT1 expression. Considering that MED16 and the precipitated DNA amount of the FRO2 and IRT1 pro- is organized in the tail module of the Mediator complex, which moters was similar to that with FIT-GFP (Fig. 8). These data acts as a bridge connecting specific transcription activators with suggest that the function of MED16 is not to modulate FIT the RNA polymerase II (Pol II) complex during transcription, we

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Table 1 Iron-deficiency response genes regulated by MED16 and FIT in roots of Arabidopsis

TPMa LogFCb

Wild-type med16-3 Wild-type med16-3/wild-type AGI ID Gene Short description +Fe Fe +Fe Fe Fe/+Fe Fe

Transcription factor AT3G12820 MYB10 myb domain protein 10 2.8 38.5 1.0 7.9 3.9 2.33 AT1G56160 MYB72 myb domain protein 72 0.2 10.9 0.2 1.1 5.3 3.23 Transporter AT4G19690 IRT1 iron-regulated transporter 1 11.4 2681.9 2.8 678.8 7.9 2.03 AT3G58060 Unknown cation efflux family protein 0.7 13.0 0.0 1.7 4.1 3.00 AT3G46900 COPT2 copper transporter 2 0.6 14.7 0.3 2.6 4.6 2.44 Other AT3G53280 CYP71B5 cytochrome p450 71b5 1.2 3.4 2.6 0.9 33.1 33.10 AT4G31940 CYP82C4 cytochrome P450, family 82 7.0 148.6 5.2 21.5 9.8 4.05 AT3G61930 Unknown molecular_function unknown 3.2 591.4 0.2 47.4 4.5 2.83 AT1G01580 FRO2 ferric reduction oxidase 2 0.2 359.6 0.0 42.8 10.8 3.10 AT1G73120 Unknown molecular_function unknown 3.0 11.0 1.5 5.5 7.6 3.68 AT1G34760 GRF11 general regulatory factor 11 6.4 63.8 8.5 29.4 3.4 1.17 AT3G50740 UGT72E1 UDP-glucosyl transferase 72E1 185.3 587.6 79.4 199.6 1.7 1.59 AT5G02780 GSTL1 glutathione transferase lambda 1 20.0 273.4 27.1 72.6 3.9 1.96 AT5G47910 RBOHD respiratory burst oxidase homologue D 51.6 121.3 19.8 50.6 1.3 1.29

Genes upregulated > two-fold in wild-type under Fe deficiency and downregulated > two-fold in med16-3 and fit mutants are shown. aTPM (transcripts per million clean tags) is the average read count for the three biological replicates in wild-type and med16-3 samples. bLogFC (fold changes) is an edgeR analysis of differential gene expression. P value < 0.05, FDR < 0.001. speculate that MED16 associates with FIT in the FIT/Ib bHLH Fe deficiency transcription activator complex and links FIT/Ib bHLH with the Pol II complex to activate downstream gene expression. In addi- tion to its bridging function, MED16 may also play a role in sta- bilizing the binding of the FIT/Ib bHLH complex to the Ib bHLH FIT promoter of FRO2 and IRT1, because the amount of precipitated DNA is c. 50% less in 35S:FIT-GFP/med16-3 than in 35S:FIT- GFP (Fig. 8). Based on previous studies and our results, we pro- pose a model whereby MED16 regulates the expression of FRO2 and IRT1 under iron limitation (Fig. 11). Pol II Mediator complex As shown in Fig. 1(b), the fit1-2 and med16 mutants dis- played similar chlorosis on half-strength MS agar plates without promoter FRO2, IRT1 iron supply. However, when grown on soil, the fit1-2 mutant Fig. 11 Schematic model for regulation of FRO2 and IRT1 expression by showed severe chlorosis and lethality (Colangelo & Guerinot, MED16 under iron deficiency in Arabidopsis. Iron deficiency signaling 2004; Jakoby et al., 2004; Yuan et al., 2005, 2008), whereas the activates/upregulates the expression of the transcriptional activator FIT med16 mutant showed only light-green coloring in the leaves and Ib subgroup bHLH (Ib bHLH) genes, including bHLH38, bHLH39, bHLH100 and bHLH101, in the cells of roots. Their products form FIT/Ib and slight growth inhibition compared with wild-type (Fig. 1c, bHLH heterodimer(s), which bind to the promoters of FRO2 and IRT1. The top section, and Fig. S1b). This discrepancy can be explained MED16 subunit of the Mediator complex, which links transcriptional by the different expression levels of the major iron uptake genes activators with the RNA polymerase II (Pol II) complex, associates with FIT IRT1 and FRO2 in the two mutants. Expression of IRT1 and in the FIT/Ib bHLH complex, stabilizes binding of the FIT/Ib bHLH FRO2 was abolished in fit1-2, but a relative amount of IRT1 complex to the promoters of FRO2 and IRT1, and transmits signals to the Pol II complex, activating expression of FRO2 and IRT1. protein accumulation (c. 30% of the wild-type, Fig. 5e,f) and FCR activity (c. 20% of wild-type; Fig. 5d) were detected in med16 mutants under iron-limited conditions. These data sug- bHLH complex with the activation of FRO2 and IRT1 expres- gest that MED16 is a major (but not unique) subunit protein sion. Such subunit(s) of the large Mediator complex will be associated with the transcription activator complex FIT/Ib detected in future studies. bHLH in the large Mediator complex that regulates FRO2 and As a co-activator, MED16 is required for lipopolysaccharide- IRT1 expression. Since no homolog of MED16 was identified induced gene expression in D. melanogaster (Kim et al., 2004). In in the Arabidopsis genome, other subunit(s) of Mediator may Arabidopsis, MED16 was originally reported to be a positive reg- have functions similar to MED16, connecting the FIT/Ib ulator of freezing tolerance and was named SENSITIVE TO

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FREEZING 6 (SFR6) (Warren et al., 1996; Knight et al., 2009). functional analysis of the STRUWWELPETER gene. EMBO Journal 21: 6036–6049. Recently, MED16 has been identified as a positive regulator of € € € the expression of circadian clock genes, such as CCA1, LHY and Backstrom S, Elfving N, Nilsson R, Wingsle G, Bjorklund S. 2007. Purification of a plant mediator from Arabidopsis thaliana identifies PFT1 as the Med25 GI (Knight et al., 2008). Additionally, it regulates the SA and JA/ subunit. Molecular Cell 26: 717–729. ET defense response (Zhang et al., 2012). In this study, we dem- Bowler C, Benvenuto G, Laflamme P, Molino D, Probst AV, Tariq M, onstrated that MED16 is involved in regulating the iron-deficient Paszkowski J. 2004. Chromatin techniques for plant cells. Plant Journal 39: – response and iron uptake. Considering that MED16 is a subunit 776 789. of the Mediator complex, which interacts with specific activator Briat JF, Fobis-Loisy I, Grignon N, Lobreaux S, Pascal N, Savino G, Thoiron S, von Wiren N, Wuytswinkel OV. 1995. Cellular and molecular aspects of iron (s) to coordinate and transfer the corresponding signal to the metabolism in plants. Biology of the Cell 84:69–81. transcriptional machinery, MED16 may act as a hub in the sig- Cerdan PD, Chory J. 2003. Regulation of flowering time by light quality. Nature naling of these pathways. It has been reported that low tempera- 423: 881–885. ture and clock circadian signals are integrated with MED16 by Chen YY, Wang Y, Shin LJ, Wu JF, Munkhtsetseg TVS, Lo JC, Chen CC, Wu affecting GI expression, which is thought to be the ‘hub’ in the SH, Yeh KC. 2013. Iron is involved in the maintenance of circadian period length in Arabidopsis. Plant Physiology 161: 1409–1420. regulation of clock and flowering time, and its expression is also Colangelo EP, Guerinot ML. 2004. The essential basic helix-loop-helix sensitive to temperature (Gould et al., 2006; Paltiel et al., 2006; protein FIT1 is required for the iron deficiency response. Plant Cell 16: Messerli et al., 2007). Recently, several studies regarding the 3400–3412. Conaway RC, Conaway JW. 2011. Function and regulation of the mediator interaction of the circadian clock with iron homeostasis have – been reported (Vert et al., 2002; Duc et al., 2009; Chen et al., complex. Current Opinion in Genetics & Development 21: 225 230. Connolly EL, Fett JP, Guerinot ML. 2002. Expression of the IRT1 metal 2013; Hong et al., 2013). Plant Fe status affects the circadian transporter is controlled by metals at the levels of transcript and protein period length by deregulating chloroplast development or the accumulation. Plant Cell 14: 1347–1357. functional state, which is a signal to the central oscillator inter- Dhawan R, Luo H, Foerster AM, Abuqamar S, Du HN, Briggs SD, preted by CCA1, LHY or GI to modulate the circadian period Mittelsten SO, Mengiste T. 2009. HISTONE length for plant growth and development (Chen et al., 2013). MONOUBIQUITINATION1 interacts with a subunit of the mediator complex and regulates defense against necrotrophic fungal pathogens in Therefore, we hypothesize that iron-limited sensitivity and the Arabidopsis. Plant Cell 21: 1000–1019. circadian phenotype may also be integrated by GI, whose expres- Doares SH, Narvaez-Vasquez J, Conconi A, Ryan C, Kidd BN, Edgar CI, sion is regulated by MED16. However, this hypothesis should be Kumar KK, Aitken EA, Schenk PM, Manners JM et al. 2009. The mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in confirmed in future studies. – In conclusion, we demonstrate that MED16, a subunit of the Arabidopsis. 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SKB1/PRMT5-mediated histone H4R3 dimethylation of Ib understanding of the molecular regulatory mechanisms of iron subgroup bHLH genes negatively regulates iron homeostasis in Arabidopsis deficiency responses and iron uptake in Arabidopsis and other thaliana. Plant Journal 77: 209–221. strategy I plants, and they reveal a key gene involved in control- Gould PD, Locke JC, Larue C, Southern MM, Davis SJ, Hanano S, Moyle ling multiple biological processes, such as freezing tolerance, iron R, Milich R, Putterill J, Millar AJ. 2006. The molecular basis of temperature compensation in the Arabidopsis circadian clock. Plant Cell 18: homeostasis, circadian clock and SA and JA/ET defense responses 1177–1187. in plant cells. These findings may be applied to molecular breed- Halliwell B, Gutteridge JMC. 1992. Biologically relevant metal ion dependent ing in crops. hydroxyl radical generation an update. FEBS Letters 307: 108–112. Henriques R, Jasik J, Klein M, Martinoia E, Feller U, Schell J, Pais MS, Koncz C. 2002. Knock-out of Arabidopsis metal transporter gene IRT1 results in iron Acknowledgements deficiency accompanied by cell differentiation defects. Plant Molecular Biology 50: 587–597. This work was supported by the National Nature Science Foun- Hong SH, Kim SA, Guerinot ML, Robertson MC. 2013. Reciprocal interaction dation of China (grant no. 31270294), the Ministry of Science of the circadian clock with the iron homeostasis network in Arabidopsis. Plant – and Technology of China (grant no. 2011CB100304), and the Physiology 161: 893 903. Challenge Program HarvestPlus-China (Agreement #8236). Jakoby M, Wang HY, Reidt W, Weisshaar B, Bauer P. 2004. FRU (BHLH029) is required for induction of iron mobilization genes in Arabidopsis thaliana. FEBS Letters 577: 528–534. References Kim TW, Kwon YJ, Kim JM, Song YH, Kim SN, Kim YJ. 2004. 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