Mitochondrial ABC Transporter ATM3 Is Essential for ... · Mitochondrial ABC Transporter ATM3 Is Essential for Cytosolic Iron-Sulfur Cluster Assembly1 Jia Zuo, Zhigeng Wu, Ying Li,

Mitochondrial ABC Transporter ATM3 Is Essential forCytosolic Iron-Sulfur Cluster Assembly1

Jia Zuo, Zhigeng Wu, Ying Li, Zedan Shen, Xiangyang Feng, Mingyong Zhang, and Hong Ye*

Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, South ChinaBotanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China (J.Z., Z.W., Y.L., Z.S., X.F., M.Z.,H.Y.); and University of the Chinese Academy of Sciences, Beijing 100049, China (J.Z., Z.W., Y.L.)

ORCID IDs: 0000-0002-5350-2456 (J.Z.); 0000-0001-7026-6240 (Z.W.); 0000-0001-5613-6458 (M.Z.); 0000-0003-3360-994X (H.Y.).

The mitochondrial ATP-binding cassette transporter ATM3 has been studied in Arabidopsis. Its function, however, is poorlyunderstood in other model plant species. This study reports that the ATM3 is required for cytosolic iron-sulfur cluster assemblyand is essential for meristem maintenance in rice (Oryza sativa). The loss of function of OsATM3 is lethal in rice at the four-leafstage. In the osatm3 T-DNA insertion mutant, the fourth leaf fails to develop and the lateral roots are short. Cytosolic iron-sulfurprotein activities were significantly reduced in both osatm3 and RNA interference transgenic lines. The expression profiles ofmany iron metabolism genes were altered in the osatm3 and RNA interference lines. Glutathione metabolism was impaired andreactive oxygen species, particularly superoxide, accumulated in osatm3. Promoter-b-glucuronidase staining of the transgenicline indicated that OsATM3 is highly expressed in lateral root primordia, root tip meristem zones, and shoot apical meristemregions. The average cell size was significantly greater in osatm3 than in the wild type. Massive cell death occurred in the osatm3root tip meristem zone. Quantitative RT-PCR revealed transcriptional reprogramming of the genes in the osatm3 and RNAi linesinvolved in DNA repair and cell cycle arrest. Our results suggest that the mitochondrial ATM3 is essential for iron homeostasisin rice.

Iron-sulfur (Fe-S) proteins are important for photo-synthesis, respiration, metabolism, and genome integ-rity (Balk and Pilon, 2011). The maturation of Fe-Sproteins requires the assembly of Fe-S clusters, whichtakes place in mitochondria, chloroplasts, and cytosolof plant cells, respectively. A number of Fe-S clusterassembly proteins have been identified in Arabidopsis(Arabidopsis thaliana; Balk and Pilon, 2011; Balk andSchaedler, 2014). Fe-S clusters can be assembled in mi-tochondria by the Iron-SulfurClustermachinery,whereasthey can be assembled in cytosol by the cytosolic iron-sulfur assembly (CIA) machinery. ATP-binding cassette(ABC) transporters are present inmitochondria.Many ofthese are half-transporters of the B subfamily. ABCsubfamily B member 7 (ABCB7) in animals (Pondarréet al., 2006), Atm1 in yeast (Kispal et al., 1999), and ABC

transporter ofmitochondrion 3 (ATM3; systematic name:ABCB25) in plants (Bernard et al., 2009) are required forcytosolic Fe-S cluster biosynthesis. In yeast, Atm1 de-pletion leads to defective Fe-S protein maturation in thecytosol and abnormal iron accumulation inmitochondria(Kispal et al., 1997, 1999). In human cells, the knockdownof ABCB7 leads to decreased cytosolic Fe-S cluster bio-synthesis and mitochondrial iron overload (Pondarréet al., 2006; Cavadini et al., 2007). Although completeknockout of ABCB7 is embryo-lethal in mammals, mis-sense mutations in ABCB7 cause X-linked sideroblasticanemia and cerebellar ataxia (XLSA/A) in humans(Rouault and Tong, 2008).

In plants, the ATM3 gene has been studied in Ara-bidopsis (Kushnir et al., 2001; Kim et al., 2006; Chenet al., 2007; Bernard et al., 2009; Teschner et al., 2010).The atm3-1 mutant, also called sta1, demonstratesdwarfism and chlorosis, and has altered leaf- and nu-cleus morphologies (Kushnir et al., 2001). ATM3 mayparticipate in the resistance to heavy metal toxicity.Plants overexpressing ATM3 have an enhanced resis-tance to cadmium (Kim et al., 2006). Three Atm1-likegenes, ATM1, ATM2, and ATM3, are present in Arabi-dopsis. ATM3 resembles yeast Atm1 most closely interms of Fe-S cluster biosynthesis (Bernard et al., 2009;Chen et al., 2007). The atm3 mutant plants display de-fects in root growth, chlorophyll content, and seedlingestablishment. Cytosolic Fe-S protein activities are sig-nificantly reduced in atm3 mutants whereas mito-chondrial and plastid Fe-S proteins are unaffected(Bernard et al., 2009). Strikingly, the atm3mutant plants

1 This work was funded by the 100 Talents Program award, Chi-nese Academy of Sciences, and by a grant from the Guangdong Sci-ence and Technology Department of China (no. 2015B020231009).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Hong Ye ([email protected]).

H.Y. and J.Z. planned the research; J.Z. and H.Y. designed theexperiments, and J.Z. performed most of them; Z.W., Y.L., Z.S.,X.F., and M.Z. provided technical assistance; H.Y. and M.Z. super-vised the experiments; J.Z. and H.Y. analyzed the data and wrote thearticle.

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do not display a dramatic iron homeostasis defect anddo not accumulate iron in mitochondria. Molybdenumcofactor (Moco) is a prosthetic group in xanthine de-hydrogenase (XDH) and aldehyde oxidase (AO). Theactivities of XDH and AO are significantly lower inatm3mutants (Bernard et al., 2009). Cyclic pyranopterinmonophosphate, the first intermediate in Moco bio-synthesis, accumulates in atm3 mutant mitochondria.ATM3 may, therefore, be involved in Moco biosynthe-sis (Teschner et al., 2010). Because many nuclear Fe-Sproteins are involved in DNA replication and repair,the CIAmachinery is essential for genome integrity andstability (Paul and Lill, 2015). Several CIA componentslike AE7 and MMS19 are required for DNA repair inArabidopsis (Luo et al., 2012; Han et al., 2015). A recentstudy showed that MMS19 is required for epigeneticregulation because it provides Fe-S clusters to DNAglycosylase ROS1. This enzyme prevents active genesfrom being silenced (Duan et al., 2015).ATM3 transporter substrates have been studied.

A functional study of an Atm1 ortholog of Novos-phingobium aromaticivorans (NaAtm1) revealed thatglutathione (GSH) derivatives serve as ATM3 trans-porter substrates. Because NaAtm1mediates the export

of metallated GSH complexes, it detoxifies heavymetals (Lee et al., 2014). Another study suggested thatAtm1 exports GSH-coordinated [Fe-S] (Li and Cowan,2015). When they are expressed in Lactococcus lactis,AtATM3 and ScAtm1 selectively transport either glu-tathione disulfide or glutathione trisulfide, but not re-duced glutathione. ATM3 may export glutathionepolysulfide, which may be a source of sulfide for thecytosolic Fe-S cluster assembly (Schaedler et al., 2014).

Putative Fe-S cluster assembly genes have beenidentified in rice (Oryza sativa). A few of these genes,including OsATM3, are highly responsive to abioticstresses (Liang et al., 2014). Little is known about thefunction of OsATM3 in rice. In this work, an osatm3T-DNA insertion mutant and RNA interference (RNAi)transgenic lines were characterized. The loss of functionof OsATM3 is lethal in rice. Mutant seedlings do notsurvive to the four-leaf stage. The mitochondrialOsATM3 is essential for cytosolic Fe-S protein biogen-esis and iron homeostasis. OsATM3 was highlyexpressed in lateral root primordia, root tip meristemzones, and shoot apical meristem (SAM) regions.Massive cell death was observed in the root tip meri-stem zone of the osatm3 mutant.

Figure 1. Identification of osatm3T-DNA insertion mutant and RNAitransgenic lines. A, Structure of theOsATM3 gene (Os06g03770). Blackboxes are exons; lines are introns.ATG and TAG are start and stop co-dons, respectively. The positions ofthe T-DNA insertion and the primers(LP, RP, and LB) are indicated. B,Identification of the heterozygoteand homozygote of the mutant bygenomicDNA PCR (top). Photographsofwild-type, heterozygote (osatm3+/2),and homozygote (osatm32/2) seed-lings are shown (bottom). Bar = 6 cm.C, SemiquantitativeRT-PCRofOsATM3transcripts in wild type and homozy-gous osatm3mutant.ACTIN1was usedas a control. D, Quantitative RT-PCR ofOsATM3 transcripts in thewild typeandhomozygous osatm3 mutant. ACTIN1was used as a control. Error bars repre-sent the SD of three biological replicates.E, Semiquantitative RT-PCR ofOsATM3transcripts in RNAi lines. ZH11 is thewild type; I10 and I37 are independentRNAi lines. F, Quantitative RT-PCR ofOsATM3 in RNAi lines. ACTIN1 wasused as a control.

Plant Physiol. Vol. 173, 2017 2097

ATM3-Dependent Fe-S Cluster Assembly in Rice

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RESULTS

OsATM3 Knockout Is Lethal in Rice at the Four-Leaf Stage

BLASTP comparison indicated that a single ATM3ortholog is present in the rice genome and encodesa half ABC transporter in mitochondria (locus:Os06g03770). It has 72% amino acid sequence identitywith the AtATM3 of Arabidopsis, and 50% identitywith ScAtm1 of yeast. This gene was named riceOsATM3 (Supplemental Fig. S1). Microscopy andtransient expression of the OsATM3 protein in Ara-bidopsis protoplasts demonstrated that it targetsmitochondria (Supplemental Fig. S2).

Three mutant lines were obtained with a T-DNA in-sertion at exon 2 (Fig. 1A), promoter (not shown), and59UTR (not shown), respectively. These were screened,and homozygous plants were identified. The mutant

lines with insertions at promoter and 59UTR, respec-tively, had no phenotype. The expression of theOsATM3gene in homozygotes was not affected in these lines (notshown). In contrast, very few homozygotes were iden-tified from the screening of the mutant line with an in-sertion at exon 2. Examination of the genotypes of222 offspring seedlings germinated from heterozygoteseeds revealed a 1:1.66:0.15 ratio of the wild type/heterozygotes/homozygotes. The number of homozy-goteswas far less thanpredicted. The results suggest thatmost of the homozygous mutants are either aborted orunable to germinate. The homozygous mutant seedlingsshowed a severe growth retardation phenotype (Fig. 1B).Therefore, the osatm3 mutant line with an insertion atexon 2 was used for subsequent analyses. Semiquanti-tative- and quantitative RT-PCR confirmed that theOsATM3 gene is completely knocked out in the osatm3

Figure 2. Phenotypes of osatm3 and RNAi lines. A, wild-type and osatm3 mutant seedlings were raised in hydroponic cultureafter germination and grown on 0.53 MS medium for 2 weeks (four-leaf stage). Bars = 3 cm in A to D. B, One week later, themutant withered and its growthwas completely retarded. C, Twoweeks later, themutant died, whereas the wild type continued tothrive. D, Comparison of wild-type and mutant seedlings after germination and growth on 0.53 MS medium for 2 weeks. Redarrows indicate the fourth leaves of the wild type and osatm3. E, Comparison of roots of the wild type and mutant after germi-nation and grown on 0.53MSmedium for 2weeks. Bar = 2 cm. The inset demonstrates lateral roots of thewild type and osatm3. Fand G, Quantitation of plant height and root length of 2-week-old seedlings (n = 30). Main roots were measured. H, Seedlings ofZH11 and RNAi lines germinated in water for 10 d. Bar = 3 cm. ZH11 is the wild type; I10 and I37 are independent RNAi lines. I,ZH11 and RNAi line plants at thematuration stage. Bar = 15 cm. J, Quantitation of root length of seedlings germinated inwater for10 d (n = 30). Main roots weremeasured. K, Quantitation of height of plants grown in paddy field (n = 15). Error bars represent theSD. Significance analysis was performed in comparison with the wild type using Student’s t test. *P , 0.05, **P , 0.01. osatm3:Homozygous mutant.

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mutant (Fig. 1, C andD). RNAi transgenic lineswere alsogenerated. Semiquantitative- and quantitative RT-PCRconfirmed that the expression ofOsATM3 is significantlyreduced in RNAi lines (Fig. 1, E and F).The osatm3mutant exhibited dwarfism and its fourth

leaf failed to develop (Fig. 2, A and D). Wild-typeseedlings thrived after the four-leaf stage. In contrast,osatm3 seedlings did not survive this stage and diedwithin twoweeks (Fig. 2, B andC). The osatm3 seedlingshad far fewer lateral roots than the wild type (Fig. 2E).Quantitation indicated that the average plant height androot length of osatm3 were significantly shorter thanthose of wild type (Fig. 2, F andG). The RNAi transgeniclines had slightly shorter shoots and roots thanwild type(ZH11; Fig. 2,H–K). Recomplementedmutant linesweregenerated to confirm that these phenotypes were indeedcaused by the insertional OsATM3 mutation. The com-plemented mutant lines survived the four-leaf stage andgrew normally (Fig. 3). The complete reversal of the le-thal phenotype in the complemented mutant lines indi-cated that the phenotypes in osatm3 are specificallycaused by the loss of function of OsATM3. Overall, theresults indicated that the knockout of OsATM3 is lethalin rice at the four-leaf stage.

Cytosolic Fe-S Cluster Assembly Is Impaired in osatm3Mutant and RNAi Lines

ATM3-like proteins are required for cytosolic Fe-Scluster biosynthesis in various species (Kispal et al.,

1999; Pondarré et al., 2006; Bernard et al., 2009). Todeterminewhether OsATM3 is involved in the cytosolicFe-S cluster assembly of rice, the enzyme activities ofthree cytosolic Fe-S proteins, aldehyde oxidase (AO),xanthine dehydrogenase (XDH), and aconitase (ACO),were analyzed. AO and XDH contain [2Fe-2S] clustersand Moco cofactors, whereas ACO contains a [4Fe-4S]cluster (Bernard et al., 2009). The activities of AO andXDH were significantly reduced in osatm3 and RNAilines relative to the wild type (Fig. 4A). Plant ACOconsists of one isoform in the cytosol and two in themitochondria (Fig. 4B). The activity of cytosolic ACOwas significantly reduced in the osatm3 and RNAi lineswhereas those of the mitochondrial isoforms were un-affected (Fig. 4A). The quantitative RT-PCR data indi-cated that the cytosolicACO gene expresses normally inosatm3 and RNAi lines (Fig. 4C). Analysis of other Fe-Sand Moco proteins like nitrite reductase (NiR) in chlo-roplasts and nitrate reductase (NR) in the cytosol indi-cated that Fe-S protein activities in the cytosol decreasedwhereas those in other organelles remain unaffected inthe osatm3 and RNAi lines (Fig. 4, D and E). These resultsindicate that OsATM3 is required for cytosolic Fe-Scluster assembly in rice.

Iron Metabolism Is Impaired in osatm3 Mutant andRNAi Lines

Several rice genes involved in iron metabolism areinduced by iron deficiency (Kobayashi and Nishizawa,

Figure 3. Complementation of osatm3 mutant withOsATM3. A, Comparison of the wild type and thecomplemented osatm3 mutant (osatm3_OsATM3).Bar = 15 cm. B, The osatm3 mutant transformed withempty vector (osatm3_ VC) died at the four-leaf stage.Bar = 3 cm. C, Quantitative RT-PCR of OsATM3transcripts. (n = 3).





2012). In roots, NAS,NAAT, and TOM1 are responsiblefor the biosynthesis and export of iron chelatorswhereas YSL15, IRT1, and IRO2 encode iron trans-porters and transcriptional regulators. The iron me-tabolism genes were expressed at minimum levelsunder normal (50 mM Fe) and excess (500 mM Fe) ironconcentrations. These genes were strongly inducedunder iron deficiency (0 mM Fe) in the wild type (Fig.5A). In contrast, they were constitutively expressed atlow levels in the osatm3 regardless of external ironconditions (Fig. 5A). MIT1 encodes a mitochondrialiron transporter and ferritin encodes the iron storageprotein in chloroplasts. In rice, they are repressed byiron deficiencies and induced by excesses of iron(Kobayashi and Nishizawa, 2012). The expression ofMIT1 and ferritin was up-regulated by high iron con-centrations in the wild type. In contrast, they wereconstitutively expressed at low levels in osatm3 (Fig.5A). IDEF1 and IDEF2 encode transcriptional regula-tors for iron metabolism and are constitutivelyexpressed in the wild type. In osatm3, excess iron

strongly induced both IDEF1 and IDEF2 (Fig. 5B).Relative to the wild type, the iron concentrationwas notsignificantly altered in osatm3 (Fig. 5C). Constitutiveexpression of several iron metabolism genes was alsofound in RNAi lines (Fig. 5D). In summary, the ex-pression profiles of several iron metabolism genes werealtered in both the osatm3 and the RNAi lines.

GSH Metabolism Is Impaired in osatm3 Mutant andRNAi Lines

GSH persulfide-containing complexes may be sub-strates for ATM3-like transporters (Lee et al., 2014;Schaedler et al., 2014; Srinivasan et al., 2014). Loss offunction of OsATM3 may impair GSH metabolism inrice. The expression ofGSH1was analyzed. It encodes aGlu-Cys ligase that catalyzes the first step in glutathi-one synthesis (Rawlins et al., 1995; Wachter et al., 2005).GSH1was up-regulated in osatm3 and RNAi roots (Fig.6A), so GSH biosynthesis may have been enhanced.

Figure 4. Fe-S cluster biosynthesis is impaired in osatm3 and RNAi lines. A, In-gel activity assays for AO, XDH, and ACO.Coomassie blue staining of SDS-PAGE indicated equal loading. ZH11 represents the wild type; I4, I10, and I37 are independentRNAi lines. B, Identification of mitochondrial ACO isoforms by in-gel activity assay. C, Quantitative RT-PCR analysis ofOs06g19960 (predicted to encode a cytosolic aconitase in rice; n = 3). D, Activity assay for NiR in leaf (n = 3). E, Activity assay forNR in leaf (n = 3). Significance analysis was performed in comparison with the wild type using Student’s t test. *P , 0.05. Cyt,Cytosolic ACO isoform; L, leaf; M, protein extract from wild-type mitochondrial fraction; mit, mitochondrial isoform; R, root; S,protein extract from whole wild-type seedlings.


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Glutathione disulfides accumulate in the mitochondriaof the Arabidopsis mutant atatm3 (Schaedler et al.,2014). ETHE1 is mitochondrial sulfur dioxygenase thatoxidizes persulfide and sulfide to thiosulfate and sul-fate, respectively (Holdorf et al., 2012; Krüssel et al.,2014). The expression of ETHE1 was analyzed andfound to be up-regulated in osatm3 andRNAi roots (Fig.6A). Total GSHwas enhanced in both osatm3 and one ofthe RNAi lines (Fig. 6B). Taken together, the results

suggest that mitochondrial GSH- and persulfide me-tabolism are impaired in the osatm3 and RNAi lines.

Reactive Oxygen Species, Especially Superoxide Anions,Accumulate in osatm3 Mutant

Mitochondrial ATM3-like transporters are often linkedto oxidative stress because they transport substances like

Figure 5. Expression profiles of iron metabolism genes are altered in osatm3 and RNAi lines. A, Quantitative RT-PCR analysis ofiron metabolism genes in osatm3 that are regulated by iron concentrations in the wild type (n = 3). Iron concentrations in hy-droponic culture are indicated: 0, 50, and 500 mM. B, Quantitative RT-PCR of transcriptional regulator genes, IDEF1 and IDEF2,which are constitutively expressed in thewild type (n = 3). C, Quantitation of iron concentrations in rice seedlings grown on 0.53MSmedium for 2 weeks (n = 3). D, Quantitative RT-PCR of iron metabolism genes in RNAi lines (n = 3). Significance analysis wasperformed in comparison with wild type using Student’s t test. *P , 0.05, **P , 0.01. L: leaf; R: root.





GSHpersulfides and heavymetals. The loss of function ofOsATM3 may cause oxidative stress in rice. The levels ofreactive oxygen species (ROS) were analyzed in theosatm3mutant. Significant amounts of superoxide anionsbut not peroxides accumulated in osatm3 leaves (Fig. 7, A,B, D, and E). Callose, a polysaccharide produced in re-sponse to biotic and abiotic stresses, deposited in osatm3leaves (Fig. 7C). Total ROS in whole lysate increasednearly 4-fold in osatm3 relative to the wild type (Fig. 7F).Overall, these results suggest that there is significant ox-idative stress in osatm3.

OsATM3 Is Highly Expressed in the Meristem

Spatiotemporal OsATM3 expression was investi-gated by generating pOsATM3:GUS transgenic rice andperforming b-glucuronidase (GUS) staining on itsseedlings. OsATM3 was initially expressed in the freshembryo after 2 d germination (Fig. 8A). It was moder-ately expressed in the coleoptile 4 d after germination(Fig. 8B).OsATM3was expressedmainly in the primaryroot tip and hypocotyl. Six d after germination, how-ever, it was strongly expressed in the coleoptile(Fig. 8C).

Many lateral roots developed along the main root atthe maturation zone. In rice seedlings at the four-leafstage,OsATM3was highly expressed in the lateral rootsbut not the main root (Fig. 8D). OsATM3 was alsohighly expressed in lateral root primordia (Fig. 8E). Theresults suggest that OsATM3 may be required for lat-eral root development. OsATM3 was also highlyexpressed in the root tip elongation- and meristemzones (Fig. 8, F and G). OsATM3 expression was gen-erally low in the shoot. It was, however, highlyexpressed in the leaf blade tip (Fig. 8H) and the basal

region of the shoot (Fig. 8I), which contain leaf pri-mordia and SAMs. The meristem consists of cells thatcan divide persistently and periodically and drive tis-sue growth and differentiation. A high expression ofOsATM3 in the root- and shoot meristems suggests thatit may be important for cell division.

Next, the spatiotemporal expression of rice OsATM3was investigated using quantitative RT-PCR (Fig. 8J). Inseeds, this gene was expressed at a minimum level. Theexpression ofOsATM3was induced at germination andgradually increased in both the shoot and the root.OsATM3 expression was high at the four-leaf stage andwas higher in the root than the shoot. At the tilleringstage,OsATM3 expression was minimal in the root andthe culm but high in the leaf blade. In the panicles, theexpression of OsATM3 was enhanced 4-fold from theP3- to the P5 stage. Therefore,OsATM3may be requiredfor seed maturation. In summary, both the GUS stain-ing and the qRT-PCR data indicate that OsATM3 par-ticipates in early seedling development (especially theroot) and that tissue-specific OsATM3 expression ishighly regulated in rice.

Root Tip Meristem Cell Death in osatm3 Mutant

It is yet unknown why the osatm3 seedlings died atthe four-leaf stage. In the attempt to determine thecause, osatm3mutant tissues were dissected at the four-leaf stage and observed with light and transmissionelectron microscopy (TEM). The average mesophyll cellsizes in the leaf blade cross sections were 11 (61.8) mmin the wild type and 22 (64.9) mm in osatm3 (Fig. 9A).The average cortex parenchyma cell sizes in the rootcross sections were 25 (63.2) mm in the wild type and31 (63.5) mm in osatm3 (Fig. 9B). TEM imaging revealed

Figure 6. GSH metabolism is impaired inosatm3 and RNAi lines. A, Quantitative RT-PCRanalysis of genes encoding Glu-Cys ligase(GSH1) and sulfur dioxygenase (ETHE1) inosatm3 and RNAi lines (n = 3). B, Total GSHquantitation of seedlings (n = 3). Significanceanalysis was performed in comparison with thewild type using Student’s t test. *P , 0.05,**P , 0.01.


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that osatm3 mesophyll cells lost their typical morphol-ogy and were significantly larger than those in the wildtype (Fig. 9C). The cell enlargement in osatm3 suggeststhat cell division is limited there. TEM also revealedthat the morphology and ultrastructure of chloroplasts(Fig. 9D) and mitochondria (Fig. 9E) seem normal butseveral osmiophilic globules were present in osatm3chloroplasts.OsATM3 is highly expressed in the root tip (Fig. 8, F

andG) and the basal shoot region (Fig. 8I). These tissuescontain the apical root and shoot meristems, respec-tively. We dissected these locations and observed themunder the microscope. The wild-type root apex crosssection included cells with large, aligned central nuclei.These are root apical meristem (RAM) cells (Fig. 9F). Incontrast, cross-section imaging revealed many deadcells and cell debris in the root apex of the osatm3mutant. These results suggest that the RAM is non-functional in osatm3 and that the death of these cellsmight be a cause of death in osatm3 seedlings. Thelongitudinal section of shoot apex demonstrated thatboth the leaf primordia (LP) and SAM are significantlyshorter in osatm3 than those in the wild type (Fig. 9G).There was no evidence of cell death in SAM. Propidium

iodine (PI) is a fluorescent nucleic acid stain. It revealsdamaged and dead cells whereas intact cells are im-permeable to it. The roots were examined using PIstaining. In contrast to the wild type, the osatm3 rootwas heavily stained by PI (Fig. 9H). Therefore, celldamage and cell death occurred in osatm3. PI stainingalso revealed that the root meristem- and elongationzones in osatm3 collapsed.

Transcriptional Reprogramming of Genes Involved inDNA Repair and Cell Cycle Arrest in osatm3 Mutant andRNAi Lines

ATM3 and the CIA pathway proteins like AE7 arerequired for the maturation of the nuclear Fe-S proteinsinvolved in DNA duplication and repair. In Arabi-dopsis, DNA repair genes (PARP1, PARP2, BRCA1,RAD51, TOS2, and GR1) are up-regulated when DNAis damaged (Luo et al., 2012). The effect of loss offunction of OsATM3 on DNA repair was analyzed us-ing the expression of the aforementioned genes. PARP2,RAD51, and TOS2 were up-regulated in osatm3 root,whereas BRCA1 and GR1 were down-regulated (Fig.

Figure 7. ROS species accumulate in osatm3mutant. A, Histochemical staining of super-oxide radicals in leaves. Bar = 1 cm. B, His-tochemical staining of H2O2 in leaves. Bar =1 cm. C, Callose deposition in osatm3 mutantrevealed by Aniline Blue staining. Bar =60 mm. D and E, Quantitation of superoxideradical spots (D) and H2O2 spots (E); n = 4. F,Total ROS measurement in whole lysates using29,79-dichlorofluorescein (n = 3). Significanceanalysis was performed in comparison withwild type using Student’s t test. *P , 0.05,**P , 0.01.





10A). Therefore, osatm3 responds abnormally to DNAdamage.

Cell division may be limited in osatm3 because itscells were larger than those in the wild type (Fig. 9). Cellcycle control genes were also analyzed. Various cyclinsand cyclin-dependent kinases (CDK) are required forG1-S and G2-M phase transitions whereas KIP-relatedprotein is a CDK inhibitor (Inzé and De Veylder, 2006;Komaki and Sugimoto, 2012). Several cyclin- and CDKgenes were down-regulated whereas KIP-related proteinwas up-regulated (Fig. 10A). Thus, there may be a cellcycle arrest in osatm3 cells. Several other genes likeCDKA;1 and CDKA;2 were up-regulated in osatm3 possi-bly in response to RAM damage. These genes are crucialfor root stem cells and meristems (Nowack et al., 2012).Several genes involved in DNA repair and cell cycle con-trol were also up-regulated in RNAi lines (Fig. 10B).

ROS and other stressors may induce programmedcell death (PCD; apoptosis) in plants. The NAC tran-scription factor, known as Os08g44820 in rice, is involvedin oxidative stress response (De Clercq et al., 2013). Mi-tochondrial heat shock protein 70 is up-regulated insalt-induced PCD of rice (Qi et al., 2011). Mitochondrialalternative oxidase 1a is induced by oxidative stress (Liet al., 2013). Upon analysis, all three genes were foundto be up-regulated in osatm3 (Fig. 10C).

DISCUSSION

The function of mitochondrial ATM3 is poorly un-derstood in plant species other than Arabidopsis. In thisstudy, our results demonstrate that ATM3 is essentialfor iron homeostasis in rice. Several iron metabolism

Figure 8. Tissue-specific expression ofOsATM3. A to I, pOsATM3:GUS expression analysis in germinating seeds and 2-week-oldtransgenic rice seedlings. A, Seed germinated for 2 d. B, Seed germinated for 4 d. C, Seed germinated for 6 d. D, Lateral roots. E,Root maturation zone. The densely stained spots are lateral root primordia. F, Root elongation zone. G, Root tip. H, Leaf blade. I,Basal region of shoot. Bars = 1mm (A–E), 2mm (F, H, and I), and 200mm (G), respectively. J, Spatiotemporal expression analysis ofOsATM3 in wild-type rice by quantitative RT-PCR (n = 3). HC, HLB, HR, andHS, Culm, leaf blade, root, and sheath at the headingstage; P1 to P5, panicles at various developmental stages as measured by the panicle length; P1, 5 cm; P2, 10 cm; P3, 18 cm; P4,23 cm; P5, mature; R3, shoot and root at the three-leaf stage; S, seed; Sh3 and R3, shoot and root at the three-leaf stage; Sh4 andR4, shoot and root at the four-leaf stage; TC, TLB, TR, and TS, culm, leaf blade, root, and sheath at the tillering stage.


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genes that are regulated by iron concentrations in wild-type rice were constitutively expressed in the osatm3and RNAi lines (Fig. 5), although this alteration is not asdramatic as the constitutive up-regulation found inyeast and mouse mutants (Kispal et al., 1999; Pondarréet al., 2006). These findings suggest that OsATM3, orthe signal exported by it, may participate in the tran-scriptional regulation of some of these genes. MIT1,which encodes the mitochondrial iron importer, andferritin, which encodes the iron storage protein in

chloroplasts, were down-regulated in osatm3 (Fig. 5A).Therefore, iron accumulation is improbable in the mi-tochondria and chloroplasts of osatm3. The resultssuggest that certain functional aspects of ATM3 inplants are indeed different from the yeast and mousehomologs.

The spatial and temporal expression ofOsATM3wasstrictly regulated in rice. It was highly up-regulatedfrom the three- to four-leaf stage in both the shoot andthe root (Fig. 8J). In rice, the four-leaf stage represents a

Figure 9. Microscopic imaging of osatm3. A, Cross section of leaf blades of wild type (left) and osatm3 mutant (right). Bars =20 mm. Eight cells were counted. B, Cross section of roots. Arrows indicate cortex thickness. Bars = 20 mm. Eight cells werecounted. C, Mesophyll cells under TEM. Bars = 2 mm. D, Chloroplasts under TEM. Arrows indicate osmiophilic globules. Bars =500 nm. E, Mitochondria under TEM. Bars = 200 nm. F, Cross section of root apex. The arrow indicates dead cells. Bars = 20 mm.G, Longitudinal section of shoot apex. Bars = 80 mm. H, PI staining of root. Bars = 200 mm. EZ, Elongation zone; MC, mesophyllcells; MTZ, maturation zone; MZ, meristem zone; VB, vascular bundles.





critical transition from the juvenile- to the adult phase.The first three leaves of the rice seedling are differen-tiated from the embryo whereas all subsequent leavesdevelop from the SAM (Poethig, 1990; Asai et al., 2002).Failure of the fourth- and subsequent leaves to developin osatm3may be explained by the dysfunction of SAM,which, in turn, may be caused by the loss of function ofOsATM3. OsATM3 was highly expressed in the lateral

roots and their primordia (Fig. 8, D and E). Therefore,OsATM3 is essential for the initiation and developmentof lateral roots in rice. In fact, the osatm3 mutant has aremarkable lateral root development phenotype (Fig.2E). OsATM3 was also highly expressed in the root tipmeristem zone (Fig. 8, F and G) wherein the RAM islocated. RAM cells divide to generate new roots. Theloss of function of OsATM3 may limit RAM cell

Figure 10. Quantitative RT-PCR analysis of genes involved in DNA repair and cell cycle control in osatm3 and RNA lines. A,Expression analysis of genes involved in DNA repair and cell cycle arrest in osatm3 seedlings (n = 3). PARP1, PARP2, BRCA1,RAD51, TOS2, and GR1 are involved in DNA repair. The other genes are involved in cell cycle arrest. B, Expression analysis ofgenes involved in DNA repair and cell cycle arrest in RNAi lines (n = 3). C, Expression analysis of genes involved in the PCD(apoptosis) of osatm3 seedlings (n = 3). Significance analysis was performed in comparison with the wild type using Student’s ttest. *P , 0.05, **P , 0.01.


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division. This inhibition may cause massive meristemzone cell death (Fig. 9F) and the collapse of the root tiparchitecture in osatm3 (Fig. 9H). The severe abnormalityidentified in the root of osatm3 impairs its nutrient up-take function and may kill it altogether. The expressionof several genes involved in DNA repair and cell cyclearrest was significantly altered in both osatm3 andRNAi lines. The changes in the gene expression, how-ever, were different between osatm3 and RNAi lines,probably because that the insertional mutation is sosevere and has secondary effects in osatm3 mutant.ABCB7, the ATM3 homolog in mammals, is required

for Fe-S protein biogenesis in the cytosol and the nuclei(Rouault and Tong, 2008; Stehling et al., 2014). Genemutations affecting a transmembrane domain inthe ABCB7 transporter cause XLSA/A in humans(Beilschmidt and Puccio, 2014; Stehling et al., 2014). Acomponent of the cytosolic Fe-S cluster assembly (CIA)such as MMS19 participates in DNA repair and ge-nomic integrity (Stehling et al., 2012). Because ABCB7acts upstream of the CIA pathway, a defect in ABCB7would probably impair the functionality of the MMS19and the CIA pathway. Therefore, an investigation intothe defects in DNA repair in XLSA/A patients might beuseful.A previous study showed that OsATM3 is an essen-

tial Fe-S cluster assembly gene involved in abiotic stresstolerance in rice (Liang et al., 2014). Its expression, es-pecially in roots, is up-regulated in response to excessiron, oxidative stress, and heavymetals. These stressorsseverely inhibit root growth (Liang et al., 2014). Thisstudy revealed thatOsATM3 is important in lateral rootdevelopment and root tip growth (Figs. 2, 8, and 9). Theresistance of rice to abiotic stress might be increased byenhancing OsATM3-mediated root development.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

The osatm3 T-DNA insertion mutants were obtained from the Crop BiotechInstitute, Kyung Hee University, Republic of Korea (Jeon et al., 2000; Jeonget al., 2006). The mutant nomenclature is Oryza sativa var Japonica ‘Dongjin’.Homozygotes were screened by sowing the seeds in soil under natural condi-tions then determining seedling genotypes by genomic DNA PCR usingprimers designed from the RiceGE database of the Salk Institute (http://signal.salk.edu/cgi-bin/RiceGE). For this study, the mutant line with a T-DNA in-sertion at exon 2 was used. Its homozygous seedling is lethal at the four-leafstage. Seeds harvested from the heterozygous mutant were germinated andgrown for 2 weeks (four-leaf stage) on 0.53 Murashige and Skoog (MS) me-dium containing 3% Suc and 0.8% agar. All rice seedlings were grown at 25 to28°C on 13-h-light/11-h-dark cycles. They were then transferred to hydroponicsolution as described in Zhang et al. (2012). Homozygous mutant seedlingswere screened and used for all subsequent analyses.

Plasmid Construction and Rice Transformation

To analyze the OsATM3 promoter, a 2167-bp promoter region upstream ofthe start codon was amplified by PCR from wild-type genomic DNA usingprimers designed from NCBI/Primer-BLAST (Supplemental Table S1). Theamplicon was subcloned upstream of uidA in pCAMBIA1391z digested withHindIII and EcoRI. To construct the RNAi vector, two 378-bp fragments of theOsATM3 coding sequence region were subcloned downstream of the Ubi-1

promoter in pTCK303 with digestion sites at KpnI/BamHI and SpeI/SacI, re-spectively.

O. sativa var Japonica ‘Zhonghua 11’ was transformed with Agrobacteriumtumefaciens strain EHA105 according to a previously published method (Hieiet al., 1997). All transgenic plants were generated on media containing25 mg$L21 hygromycin B then transferred to 0.53MS medium (supplementedwith 1.5% Suc and 0.8% agar) for rooting. The regenerated plants were grownon soil under natural conditions. The T2 or T3 generation of the homozygoustransgenic lines was screened using PCR (Supplemental Table S1).

To complement the osatm3 mutant, the OsATM3 ORF sequence wassubcloned into pCAMBIA2301 at the EcoRI and AdeI digestion sites and drivenby its native promoter (a 2-kb region upstream of the start codon). An emptyvector was used as a negative control. The construct was used to transformcallus induced from the seeds of heterozygous mutants via A. tumefaciens-mediated transformation. There were not enough homozygous seeds availablefor callus induction. G418 disulfate salt (Sigma-Aldrich) was used as a selectionmarker. Positive plants with a homozygous mutant background were screenedfrom the T1 or T2 generation by PCR.

Subcellular Localization of OsATM3

The full-length coding region (2,199 bp without stop codon) or the signalpeptide region (294 bp) ofOsATM3was amplified by PCR (Supplemental TableS1). The PCR amplicon was digested with SalI and SacI and fused to a p-35S-GFP vector driven by aCaMV 35S promoter. The resulting constructs were usedfor transformation and transient expression in Arabidopsis protoplasts viapolyethylene glycol-mediated transformation as described in Yoo et al. (2007).Mt-rk was used as a mitochondrial marker. It has a yeast (Saccharomyces cer-evisiae) cytochrome oxidase IVmitochondrial targeting sequence fused with thered fluorescent protein mCherry. The transformed protoplasts were viewedunder a fluorescence microscope (Axio Imager A2; Carl Zeiss).

Semiquantitative RT-PCR and Quantitative RT-PCR

Total RNA was extracted from rice tissues using Plant RNA Kit (Omega)following the manufacturer’s instructions. The extracted RNA was reverse-transcribed into cDNA using a reverse transcription kit including a genomicDNA eraser (TaKaRa). Semiquantitative RT-PCR was performed in 25 or28 cycles for ACTIN1 and other genes and 35 cycles for OsATM3. ACTIN1(Os03g50890) was used as the internal control. Quantitative RT-PCR was con-ducted in 40 cycles on a Light Cycler 480II (Roche) according to the manufac-turer’s manual (SYBR Premix Ex Taq; TaKaRa). Primer sequences are shown inSupplemental Table S1.

Enzyme Activity Assays

Proteins were extracted by grinding leaf or rot tissues with equal volumes ofextraction buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1% [v/v] Triton X-100,1 mM DTT, Roche Protease Inhibitor Cocktail, and 1 mM PMSF), followed bycentrifugation at 12,000 rpm, 4°C for 30 min. Protein was quantified usingProtein Assay Kit II (Bio-Rad). For the ACO, AO, and XDH activity assays, 50 to100 mg protein was mixed with loading buffer (20 mM Tris-HCl 8.0, 80% [v/v]glycerol, and 0.1% [w/v] Bromophenol Blue) and then separated on nativePAGE gel. The details of in-gel activity assays for ACO, AO, and XDH weredescribed previously (Koshiba et al., 1996; Bernard et al., 2009). NR activity wasmeasured based on nitrite production. NiR activitywas assayed as described byTakahashi et al. (2001). Two-hundred microliters of protein extract was mixedwith 800 mL of a solution containing 100 mM P buffer at pH 7.5, 75 mM KNO3,and 0.5 mg$mL21 NADH incubated at 25°C for 30 min. Immediately after in-cubation, 1 mL of 1% (w/v) sulfanilamide dissolved in 3 M HCl and 1 mL of0.02% (w/v) N-(1-naphthyl)ethylenediamine dihydrochloride were added tothemixture that was then left to stand 15min after which its optical density wasmeasured in a spectrophotometer at 540 nm.

Mitochondrial Fractionation

Mitochondria-enriched fractions were prepared from hydroponically cul-tured rice seedlings following the method of Sweetlove et al. (2007). Approxi-mately 10 g rice seedlings were homogenized on ice with 50 mL extractionbuffer containing 0.3 M Suc, 25 mM Na4P2O7, 2 mM EDTA, 10 mM KH2PO4, 1%(w/v) PVP-40, 1% (w/v) BSA, and 20 mM ascorbic acid at pH 7.5. The extract




http://signal.salk.edu/cgi-bin/RiceGE

http://signal.salk.edu/cgi-bin/RiceGE







was filtered through Miracloth (Merck Millipore). The filtrate was centrifugedat 1,100g for 5 min at 4°C. The resulting supernatant was centrifuged at 18,000gfor 20 min at 4°C. Pellets were collected as mitochondria-enriched fractions.

Iron Quantification and GSH Measurement

Iron concentrations in rice tissues were quantified by inductively coupledplasma mass spectroscopy (7700 series; Agilent Technologies) as described byLiang et al. (2014). The GSH content was measured as described by Kim et al.(2006) but with modifications. Whole plants were ground in 5% sulfosalicylicacid at 4°C then centrifuged twice at 14,000g for 15 min at 4°C. The supernatantwas diluted in 0.1 M P buffer (pH 7.4), of which 2.48 mL of the diluted super-natant was mixed with 20 mL of 10 mM 59,59-dithiobis (2-nitrobenzoic acid) andincubated for 5 min. Absorbance was measured at 412 nm. The GSH contentwas calculated using a GSH standard curve.

Detection of ROS

ROSwere detected by histochemical staining and fluorospectrophotometry.Superoxide anion was stained with NBT. Hydrogen peroxide was stained withdiaminobenzidine tetrahydrochloride. Leaf fragments of equal size were se-lected and stained to enumerateNBT anddiaminobenzidine tetrahydrochloridespots. Statistical analyses were applied to the spot counts. To determine totalROS, whole plants were ground in liquid nitrogen, dissolved in 10mM Tris-HCl(pH 7.2), and centrifuged at 12,000g for 20 min at 4°C. The supernatant wasmixed with esterified 29,79-dichlorofluorescein and its fluorescence monitoredin an LS 55 Fluorescence Spectrometer (Perkin-Elmer). Callose deposition wasvisualized by Aniline Blue staining (Wang and Liu, 2006) and observed under afluorescence microscope (Axio Imager A2; Carl Zeiss).

b-GUS Staining

T2 or T3 generation plants were used for GUS staining as described else-where, but with modifications (Jefferson et al., 1987). Tissues were sampled andincubated in 50 mM sodium P buffer (pH 7.0) containing 1 mM X-gluc, 1 mM

EDTA, 0.05% (v/v) Triton X-100, 0.1 mM potassium ferrocyanide, and 0.1 mM

potassium ferricyanide at 37°C overnight, then transferred into 70% (w/v)ethanol to remove the chlorophyll. Stained tissues were imaged with a M165Cstereomicroscope (Leica).

Sections and Microscopy Observation

Leaves or roots from 2-week-old rice seedlings were fixed in 4% parafor-maldehyde (Sigma-Aldrich) and 2% glutaraldehyde (Sigma-Aldrich) in 0.1 M

PBS (pH 7.2) and postfixed in 2% (w/v) osmium tetroxide in 0.1 M PBS over-night. The fixed specimens were embedded in SPI-Pon 812 (SPI Supplies), andused to make ultrathin (80 nm) or semithin (2 mm) sections with an Ultracut Eultramicrotome (Leica). TEM was performed following the manufacturer’smanual. Images were photographed using a JEM-1010 Electron Microscope(JEOL) at 15 kV. Basal region of the 2-week-old rice seedling was fixed in 70%ethanol, 5% acetic acid, and 3.7% formaldehyde for 24 h, and dehydratedthrough a graded ethanol series. After vitrification by dimethylbenzene,samples were embedded in paraffin and sectioned on a RM2255 microtome(Leica). The 8- to 10-mm-thick sections were attached to adhesion slides(positively charged) stained with Toluidine Blue or sarranine and viewedwith an Axio Imager A2 (Carl Zeiss). Root tips were stained with 5 mg/Lpropidium iodide dissolved in sterile water for 20 min, treated with chloralhydrate, and observed under an LSM-510 confocal laser scanning microscope(Carl Zeiss).

Statistics

Significance was determined using Student’s t test in the software SPSS(IBM). P , 0.05 was considered significant.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Amino acid sequence alignment of ScAtm1p,AtATM3, and OsATM3.

Supplemental Figure S2. Subcellular localization of OsATM3 in Arabidopsisprotoplasts OsATM3 and OsATM3SP indicate the full-length proteinand the signal peptide (98 amino acid residues from the N terminus aspredicted by TargetP v. 1.1, http://www.cbs.dtu.dk/services/TargetP/),respectively.

Supplemental Figure S3. Semiquantitative RT-PCR analysis of iron me-tabolism genes in osatm3.

Supplemental Figure S4. Semiquantitative RT-PCR analysis of iron me-tabolism genes in RNAi lines.

Supplemental Figure S5. Semiquantitative RT-PCR analysis of GSH1 andETHE1 in osatm3 and RNAi plants.

Supplemental Table S1. Primers used in this study.

ACKNOWLEDGMENTS

We thank Peiwei Liu, Meihuan Wang, Kuaifei Xia, Lu Qin, and Hui Mo fortechnical assistance and Rufang Deng for transmission electron microscopy.

Received November 16, 2016; accepted February 28, 2017; published March 1,2017.

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