down-regulation of a silent information -related ... meiosis (for review, see blander and guarente,...

Down-Regulation of a SILENT INFORMATIONREGULATOR2-Related Histone Deacetylase Gene, OsSRT1,Induces DNA Fragmentation and Cell Death in Rice1[C][W]

Limin Huang2, Qianwen Sun2, Fujun Qin, Chen Li, Yu Zhao, and Dao-Xiu Zhou*

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University,Wuhan 430070, China (L.H., Q.S., F.Q., C.L., Y.Z.); Department of Quartermaster,Military Economy Academy, Wuhan 430035, China (L.H.); and Institut de Biotechnologiedes Plantes, Universite Paris Sud 11, 91405 Orsay, France (D.-X.Z.)

The SILENT INFORMATION REGULATOR2 (SIR2) family proteins are NAD1-dependent histone deacetylases. Sir2 isinvolved in chromatin silencing at the mating-type loci, rDNA, and telomeres in yeast and is associated with lifespan extensionin yeast, worms, and flies, but also in a broader range of additional functions. In this work, we investigated the role of OsSRT1,one of the two SIR2-related genes found in rice (Oryza sativa). We show that OsSRT1 is a widely expressed nuclear protein withhigher levels in rapidly dividing tissues. OsSRT1 RNA interference induced an increase of histone H3K9 (lysine-9 of H3)acetylation and a decrease of H3K9 dimethylation, leading to H2O2 production, DNA fragmentation, cell death, and lesionsmimicking plant hypersensitive responses during incompatible interactions with pathogens, whereas overexpression ofOsSRT1 enhanced tolerance to oxidative stress. Transcript microarray analysis revealed that the transcription of many trans-posons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was ac-tivated. Chromatin immunoprecipitation assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation onthe transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be amongthe primary targets of deacetylation regulated by OsSRT1. Our data together suggest that the rice SIR2-like gene is required forsafeguard against genome instability and cell damage to ensure plant cell growth, but likely implicates different molecularmechanisms than yeast and animal homologs.

Histone acetylation involves the transfer of acetylgroups from acetyl-CoA to Lys residues of histones.Hyperacetylation of histones leads to relaxation of chro-matin structure and is associated with transcriptionalactivation, whereas hypoacetylation of histones induceschromatin compaction and gene repression (Carrozzaet al., 2003). Histone acetylation is catalyzed by histoneacetyltransferases, whereas histone deacetylation is cat-alyzed by histone deacetylases (HDACs). Plant HDACscan be grouped into four subclasses. Three of themhave primary homology to the three classes of HDACs(RDP3, HDA1, and SIR2) found in yeast and animalcells (Pandey et al., 2002). The fourth class of plantHDACs (known as the HD2 class) is found only inplants (Lusser et al., 1997; Pandey et al., 2002).

The SILENT INFORMATION REGULATOR2 (SIR2)family proteins, known also as sirtuins, are NAD1-de-pendent protein deacetylases. They contain a 200-aminoacid domain (Pfam designation PF02146) conservedfrom bacteria to humans (Frye, 2000). Based on vari-ations in this domain, the eukaryotic SIR2 proteins fallinto four main classes (Frye, 2000). Yeast SIR2 belongsto class I of sirtuin genes and is involved in chromatinsilencing, DNA repair, and chromosome fidelity dur-ing meiosis (for review, see Blander and Guarente,2004). Deletion of yeast SIR2 leads to histone H3 andhistone H4 hyperacetylation of subtelomeric regions,the mating-type loci, and the rDNA loci (Robyr et al.,2002). Sir2-related proteins have been implicated inmediating lifespan increases in yeast, worms, and flies,but also in a broader range of additional functions (forreview, see Blander and Guarente, 2004; Haigis andGuarente, 2006).

Yeast has four additional Sir2 homologs, termed Hst1to Hst4, in addition to the founding member. All of theyeast members belong to class I of the Sir2-related pro-teins (Frye, 2000). Mammalian cells have seven mem-bers of the SIR2 family (SIRT1–SIRT7), distributed intoall four classes (Frye, 2000). Three of the mammalianmembers are localized in the nucleus; the remainingmembers are either cytoplasmic or mitochondrial lo-calized (for review, see Haigis and Guarente, 2006).

Plant genomes seem to contain relatively fewer SIR2homologs than the other eukaryotes. In Arabidopsis

1 This work was supported by grants from the National SpecialKey Program of Rice Functional Genomics and the National NaturalScience Foundation of China.

2 These authors contributed equally to the article.* Corresponding author; e-mail [email protected]; fax 33–1–

69153424.The author responsible for distribution of materials integral to

the findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Dao-Xiu Zhou ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.107.099473

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(Arabidopsis thaliana), only two SIR2 family gene se-quences (named atSRT1 and atSRT2) have been iden-tified. Phylogenetic analysis of identified plant SIR2homologs shows that they belong to only two of thefour classes of the family, classes that have only plantand animal members (Pandey et al., 2002; Fig. 1). So farno physiological function has been assigned to plantSir2-related proteins. As there are fewer SIR2-relatedgenes found in plant genomes, important questionsarise, such as whether plant Sir2-related proteins con-serve similar functions as yeast and animal homologs.In this work, we studied the function of a rice (Oryzasativa) SIR2-like gene, OsSRT1 (also called OsSIRT701;Pandey et al., 2002), by transgenic approaches. Ourdata show that OsSRT1 was preferentially expressed inrapidly dividing young tissues/organs and the proteinwas nuclear localized. Phenotypic and molecular anal-ysis of RNA interference (RNAi) transgenic plants sug-gests that OsSRT1 is involved in H3K9 (Lys-9 of H3)deacetylation required for transcriptional repression

of transposable elements and apoptosis-related genes.Our data suggest that OsSRT1 may have a function inthe safeguard against genome instability and DNA dam-age to ensure plant cell growth.

RESULTS

Rice Genome Contains Two SIR2-Related Genes

Sequence analysis of the rice genome revealed twoSIR2-related genes, named OsSRT1 and OsSRT2. OsSRT1and other plant SRT1 homologs are found in the sameclass (class IV), whereas OsSRT2 belongs to class II ofthe SIR2-related genes (Pandey et al., 2002; Fig. 1).There were no plant members found in class I and classIII of the SIR2 family. Plant predicted SRT1 proteinsshowed relatively high conservation. Only the N-terminalparts of the plant proteins were conserved with theanimal homologs (data not shown). Northern-blot anal-ysis revealed that OsSRT1 was generally expressed indifferent tested rice tissues, but with higher transcriptlevels detected in tissues with high cell proliferationrates, such as buds, seedlings, and developing panicles(Fig. 2A). The animal members of class IV proteins, suchas human HsSIRT6 and HsSIRT7, are nuclear localized.To detect the subcellular localization of OsSRT1, thecoding region of the cDNAwas fused to the GFP-codingsequence under the control of the maize (Zea mays)ubiquitin promoter and transiently transfected intoonion (Allium cepa) cells. The fusion protein was local-ized in the nucleus (Fig. 2B).

Down-Regulation of OsSRT1 by RNAi InducedProgrammed Cell Death in Rice

To study the physiological function of OsSRT1, a412-bp segment of the 3#-untranslated region of thegene (Fig. 3A), which was not conserved with OsSRT2,was inserted in inverted repeats to build a constructfor RNAi. The construct was used to transform an indicarice variety ‘Minghui63’. About 20 independent trans-genic lines were produced and analyzed for OsSRT1 ex-pression during the root regeneration stage. Three ofthem showed either reduced or no expression of theendogenous gene, suggesting an effect of RNAi (Fig.3B). To further analyze whether there was any effectof OsSRT1 RNAi on histone modification, we didwestern-blot analyses using antibodies raised specifi-cally against acetylated histone H3 and acetylated H3K9,because several nuclear SIR2 proteins in yeast and animalcells have been shown to be mainly involved in histoneH3 and H3K9 deacetylation (Blander and Guarente,2004). As H3K9 dimethylation is closely associated withH3K9 deacetylation (Strahl and Allis, 2000), we alsotested with antibodies against dimethylated H3K9.As shown in Figure 3C, the OsSRT1 RNAi had littleeffect on overall histone H3 acetylation. However, theacetylation of H3K9 was induced, whereas the dimeth-ylation of H3K9 was reduced, in agreement with theantagonistic relationship between H3K9 acetylation

Figure 1. Neighbor-joining tree of SIR2-related proteins from eukaryotes.Abbreviations are as follows (in parentheses): Arabidopsis (at), Caeno-rhabditis elegans (ce), Drosophila melanogaster (dm), Homo sapiens(hs), rice (os), Saccharomyces cerevisiae (sc), Schizosaccharomycespombe (sp), wheat (ta), and maize (zm). Four subclasses are indicated.

Rice SIR2-Related Gene Function

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and dimethylation. Transgenic lines (7R-16 and 7R-17)with no reduction of OsSRT1 transcripts (Fig. 3B) showedno alteration in the histone modifications (Fig. 3C), sug-gesting that the phenotype was induced by OsSRT1down-regulation.

The RNAi lines were selected for phenotype obser-vation and further analysis. The RNAi plants at thetwo-leaf stage (about 14 d after germination) began toproduce brown dots on leaves, which became larger atlatter stages, leading to precocious leaf senescence(Fig. 4). Only two of the three RNAi lines could pro-duce seeds. The severest line died before getting intomaturity. The transgenic lines showing no alteration ofOsSRT1 expression or histone modification did not man-ifest the phenotype (data not shown), suggesting thatthe lesion mimic phenotype was induced by OsSRT1down-regulation. The lesions were reminiscent of celldeath induced by hypersensitive responses during plantpathogen infections, suggesting that OsSRT1 RNAimight have induced programmed cell death (PCD). Totest this hypothesis, young leaf sheaths (T1 generation)were incubated with 3,3#-diaminobenzidine (DAB) todetect H2O2 (Thordal-Christensen et al., 1997). H2O2production was detected in cells of OsSRT1 RNAi leafsheaths at day 7 after germination (before appearanceof the symptom). At day 21 after germination, morecells produced H2O2 at higher levels (Fig. 5A). H2O2 pro-duction is an indicator of plant cells undergoing hy-

persensitive PCD during incompatible plant-pathogeninteractions (Brodersen et al., 2002). Therefore, thelesion mimic phenotype of OsSRT1 RNAi plants sug-gested that the down-regulation of OsSRT1 inducedPCD in rice. To confirm whether OsSRT1 RNAi in-duced PCD, the second leaves of 2-week-old wild-typeand OsSRT1 RNAi (T1 generation) plants were fixed,sectioned, and processed for terminal deoxyribonu-cleotidyl transferase-mediated dUTP nick-end labeling(TUNEL). TUNEL can sensitively detect DNA frag-mentation, one of the hallmarks of PCD, by labelingexposed 3# hydroxyl ends of DNA fragments usingfluorescein-dUTP. The same leaf sections were simul-taneously stained with propidium iodide to reveal allnuclei (red) in each section. None of the nuclei in wildtype was TUNEL positive but, in contrast, most nucleiin the OsSRT1 RNAi leaf section were TUNEL positive(green; Fig. 5B), indicating that DNA damage wasgenerally induced in the OsSRT1 RNAi leaves.

Overexpression of OsSRT1 Enhanced Tolerance toOxidative Stress

To further study the function of OsSRT1, the cDNAwas inserted in an overexpression vector under theFigure 2. Expression profiles of OsSRT1. A, Northern hybridization

detection of OsSRT1 mRNA in different rice tissues or developmentalstages. Actin transcripts were detected as controls. B, Nuclear localiza-tion of the OsSRT1-GFP fusion. Top, Onion skin cells transfected withOsSRT1-GFP photographed under a confocal microscope at 488 nm (left)and merged with the transmission image (right). Bottom, GFP alone.Bars 5 40 mm. [See online article for color version of this figure.]

Figure 3. OsSRT1 RNAi affected overall H3K9 acetylation and dimethyl-ation. A, Schematic representation of the OsSRT1 cDNA. The coding regionis boxed. The dark region corresponds to the conserved catalytic domain.TheDNAsegmentused to construct theRNAivector is indicated.B, RT-PCRanalysis of OsSRT1 transcripts in the wild type and three transgenic lines(LM-1–LM-3).Acomparisonbetween thewild typeand twononphenotypictransgenic lines (7R-16 and 7R-17) is shown on the right. Actin transcriptswere detected as controls. C, Western-blot analysis of enriched histonefractions isolated from the wild type and pooled transgenic 7R or LM linesshown inBwithantibodies against acetylatedhistoneH3,acetylatedH3K9,and dimethylated H3K9, as indicated. WT, Wild type.

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control of the maize ubiquitin promoter. More than 30independent lines were obtained. Northern-blot anal-ysis showed that most of the transgenic plants over-expressed OsSRT1 (Fig. 6A). Western-blot analysis ofenriched histone fractions detected a decrease of H3K9acetylation in overexpression plants (Fig. 6B). Theoverexpression plants showed no particular visibleor morphological phenotype. However, when treatedwith paraquat (1,1#-dimethyl-4,4#-bipyridylium), anherbicide that induces oxidative stresses in plants, theoverexpression plants showed an enhanced tolerancecompared to the wild type, as demonstrated by fewerand smaller lesions observed on the overexpressionplants than the wild type (Fig. 6, C and D), whereas noclear difference was seen between wild-type and trans-genic siblings without overexpression of OsSRT1 (Sup-plemental Fig. S1). This suggests that the increasedtolerance to paraquat was induced by OsSRT1 over-expression.

Transcriptomic Analysis Revealed Activation of Many

Transposon and PCD-Related Genes

To study whether the down-regulation of OsSRT1affected gene expression, we compared the transcriptsof the RNAi to the wild-type plants by microarray anal-ysis (Affymetrix). RNAs were isolated from young

leaves of 11-d-old plants (before appearance of lesionsin the RNAi plants). Analysis of data from three bio-logical repeats revealed that 521 genes were up-regulatedand 213 down-regulated (with q value at 5%). (Thedata are under the accession no. GSE7197 at http://www.ncbi.nlm.nih.gov/geo.) Gene ontology classifi-cation of the deregulated genes revealed that mostcategories had more up- than down-regulated genes,in agreement with the global up/down ratio (Table I).For instance, the transposon categories (both DNA andretroelements) had 40 members activated in the RNAileaves, but only four DNA elements were repressed.Most of the activated transposons listed in Table IIwere not expressed in wild-type rice as revealed bybackground signals of the microarray hybridizations. Inaddition, a relatively large number of stress-responsiveand stress-related (i.e. phenylpropanoid metabolism,defense response, DNA repair) genes were deregulated(Table I). To confirm the microarray data, we performedsemiquantitative reverse transcription (RT)-PCR analy-sis of RNA isolated from OsSRT1 RNAi young leavesharvested at 7, 11, and 21 d after germination tocompare with wild-type and OsSRT1 overexpressionplants. As shown in Figure 7A, the expression of tested

Figure 4. OsSRT1 RNAi induced a lesion mimic phenotype. A, AnOsSRT1 RNAi seedling at two-leaf stage (right) compared to a wild-typeplant at the same age (left), showing that the development of lesionsaffected the growth. Bar 5 3 cm. B, Enlarged view of the RNAi plants inA. Bar 5 0.6 cm. C, An OsSRT1 RNAi plant at three-leaf stage (right)compared to a wild-type plant at the same age (left). Bar 5 3 cm. D,Enlarged view of a leaf from the RNAi plant shown in C. Bar 5 0.5 cm.E, Comparison of an RNAi plant (right) with the wild type (left) attillering stage. Bar 5 16 cm. F, Comparison of an RNAi plant (right) withthe wild type (left) at mature stage. Bar 5 20 cm. G, Enlarged views ofleaves of the RNAi plant at tillering stages. Bar 5 3 cm.

Figure 5. OsSRT1 RNAi induced H2O2 production and genomic DNAfragmentation. A, DAB detection of H2O2 in OsSRT1 RNAi (a–d) andwild-type (e–h) plants at day 7 (a, e, c, and g) and day 21 (b, f, d, and h)after germination. Sections from leaf blades (a, b, e, and f) and fromsheaths (c, d, g, and h) are shown. Bar 5 20 mm. B, Detection of nuclearDNA fragmentation by in situ TUNEL assay. In situ TUNEL assay fordetection of DNA cleavage was performed using young leaf tissues atday 21 after germination. The upper fifth of the leaf was used for assay.All the cross sections were counter-stained with propidium iodide. aand c are the corresponding negative controls for b and d, respectively.a and b are the leaves of the wild type, and c and d are the leaves ofOsSRT1 RNAi plants. Bar 5 28 mm.





DNA and retroelements was induced early in 7-d-oldRNAi leaves. Overexpression of OsSRT1 had a nega-tive effect on the expression of the DNA elements, butseemed to have some positive effect on the two testedretroelements (Fig. 7A). It was not known at this stagewhether the induction of the retroelement was directlyrelated to the OsSRT1 overexpression or a consequenceof an indirect effect induced by the overexpression.

We compared the expression of two hypersensitiveresponse (HSR201 and HSR203J) marker genes (Czernicet al., 1996) and a cytochrome P450 gene (called APO) thatis closely related to wheat (Triticum aestivum) CYP709C1and CYP709C3v2, both of which are suggested to beinvolved in wheat defense to pathogens (Kandel et al.,2005; Kong et al., 2005). HSR201 and APO, but notHSR203J, were found to be induced by OsSRT1 RNAiin the microarray data (Table III). Consistently, theRT-PCR results showed that HSR201 and APO, but notHSR203J, were activated early in 7-d-old RNAi plants(Fig. 7B). In contrast, HSR203J was repressed by OsSRT1overexpression (Fig. 7B).

The microarray data revealed that the OsSRT1 RNAiinduced SAG12 but not SAG13 (Table III), both of whichare senescence-associated genes (Pontier et al., 1999;Brodersen et al., 2002). RT-PCR revealed that SAG12

was induced 11 d after germination. OsSRT1 over-expression repressed the SAG12 at day 21 after germi-nation. The expression of SAG13 was not significantlyaltered by the deregulation of OsSRT1 (Fig. 7B). Themicroarray data also showed moderate induction ofPATHOGENESIS-RELATED (PR) genes that are acti-vated during hypersensitive responses (Table III). TheRT-PCR results showed the induction of the tested PRgenes 11 d after germination, later than that of HSR201and APO (Fig. 7B), in agreement with the fact that PRgene induction is a downstream event of hypersensi-tive responses. OsSRT1 overexpression showed induc-tion of PR1a and PR1b at day 7 after germination, butrepression of the tested PR genes at day 21 after ger-mination. These observations suggested that PR genesmight be not the direct targets of OsSRT1 and that thederegulation of OsSRT1 might have an indirect impacton PR gene expression.

OsSRT1 RNAi Induced Histone H3K9 Acetylation onTransposable Elements

To study whether the activation of the transposableelements and PCD marker gene expression by down-regulation of OsSRT1 was linked to alterations in histoneacetylation, we performed chromatin immunoprecipi-tation (ChIP) assays. Chromatin fragments isolated from11-d-old leaves of wild-type and OsSRT1 RNAi plants

Figure 6. Overexpression of OsSRT1 conferred tolerance to paraquattreatment. A, Northern-blot analysis of OsSRT1 overexpression indifferent transgenic lines compared to the wild type. The 18S ribosomalRNA levels were revealed as controls. B, Western-blot analysis ofenriched histone fractions from pooled samples of the overexpressionplants with antibodies against acetylated histone H3K9. C, Comparisonof overexpression plants with wild-type ones challenged by 10 mM

paraquat. D, Comparison of leaves from overexpression and wild typetreated with or without 10 mM paraquat. WT, Wild type; Ox, over-expression plants. [See online article for color version of this figure.]

Table I. Gene ontology categories of up-regulated (Up) anddown-regulated (Down) genes found by Affymetrix microarrayanalysis of OsSRT1 RNAi plants compared to the wild type

Gene Ontology Nos. Gene Ontology Annotation Down Up

GO:0003700 Transcription factor activity 16 30GO:0006281 DNA repair 0 2GO:0006313 DNA transposition 4 22GO:0032197 Transposition, RNA mediated 0 18GO:0012501 PCD 2 7GO:0045454 Cell homeostasis 0 5GO:0007165 Signal transduction 23 27GO:0006629 Lipid metabolism 21 23GO:0006810 Transport 28 53GO:0009698 Phenylpropanoid metabolism 5 18GO:0015979 Photosynthesis 2 1GO:0019538 Protein metabolism 35 62GO:0007049 Cell cycle 0 7GO:0005975 Carbohydrate metabolism 7 13GO:0006952 Defense response 24 41GO:0016043 Cell organization and biogenesis 25 50GO:0006139 Nucleobase, nucleoside,

nucleotide, and nucleicacid metabolism

21 60

GO:0007275 Development 24 60GO:0006519 Amino acid and derivative

metabolism10 35

GO:0006950 Response to stress 44 84GO:0040007 Growth 11 14GO:0007568 Aging 5 2Unknown Unknown 143 275Total 313 521

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Table II. Transposon elements induced by OsSRT1 RNAi

Transposon Class Family Locus Affymetrix Probe Sets Log2

Class I (retrotransposons) Ty3-gypsy subclass retrotransposon family Os03g19600 Os.8570.3.S1_s_at 4Os03g50670 Os.48948.1.S1_x_at 5.6Os02g32880 Os.46692.1.S1_at 5.4Os09g31930 Os.52004.1.S1_at 2.8Os03g50670 Os.48948.1.S1_x_at 5.6Os04g07770 Os.41695.3.S1_x_at 1.9Os10g08340 OsAffx.8464.1.S1_at 3.6

Ty1-copia subclass retrotransposon family Os08g03880 Os.23300.1.S1_at 4Non-LTR retrotransposon family (LINE) Os12g41440 Os.20851.1.A1_x_at 2.9

Os05g26730 Os.20851.1.A1_x_at 2.9Unclassified Os04g48650 Os.20851.1.A1_x_at 2.9

Os05g26740 Os.20851.1.A1_x_at 2.9Os08g22520 Os.20851.1.A1_x_at 2.9Os03g33160 Os.20851.1.A1_x_at 2.9Os07g09760 OsAffx.8923.1.S1_at 2.5Os05g30290 OsAffx.8923.1.S1_at 2.5Os04g51870 OsAffx.8923.1.S1_at 2.5Os03g59190 OsAffx.8923.1.S1_at 2.5Os11g36110 OsAffx.8923.1.S1_at 2.5Os11g29950 OsAffx.8923.1.S1_at 2.5Os07g05440 Os.50940.1.S1_a_at 5.3Os08g10250 Os.52458.1.S2_at 1.6Os03g18880 Os.53278.1.S1_at 4.5Os02g14210 OsAffx.24244.1.S1_at 3.9Os08g35020 Os.50495.1.S1_at 5.5Os07g44720 Os.51357.1.S1_at 1.4Os07g31690 Os.51357.1.S1_at 1.4Os07g23980 Os.26542.1.A1_at 1.7Os07g20260 Os.26542.1.A1_at 1.7

Class II (DNA transposons) hAT-like transposase family Os03g36550 Os.23305.1.A1_at 6Os06g11830 Os.20614.3.S1_x_at 9Os07g15340 Os.20614.3.S1_x_at 9Os11g40360 OsAffx.424.1.S1_at 8.4Os07g47800 OsAffx.424.1.S1_at 8.4Os10g02910 OsAffx.424.1.S1_at 8.4

Pong subclass transposase family Os10g31120 OsAffx.8070.1.S1_x_at 5.3Os10g41350 OsAffx.8070.1.S1_x_at 5.3Os01g60590 OsAffx.8070.1.S1_x_at 5.3Os01g65240 Os.31886.1.S1_at 2.8Os01g60580 OsAffx.21778.2.S1_at 5.9Os01g27580 OsAffx.8898.1.S1_at 2.1Os12g12500 OsAffx.8898.1.S1_at 2.1Os05g43630 OsAffx.8898.1.S1_at 2.1

CACTA, En/Spm subclass transposase family Os01g60530 Os.8248.2.S1_x_at 7Os10g30670 Os.47371.1.S1_at 5.4Os10g26910 Os.48948.1.S1_x_at 4.1Os10g21980 Os.46692.1.S1_at 7.6Os04g29620 Os.46692.1.S1_at 7.6Os11g22240 Os.46692.1.S1_at 7.6Os07g24940 Os.6645.1.S1_s_at 3.4Os07g24960 Os.6645.1.S1_s_at 3.4Os08g34390 Os.56875.1.S1_at 4.4Os07g32710 OsAffx.22999.1.S1_at 3.1Os10g26910 OsAffx.26155.1.S1_x_at 3.7

Mutator subclass transposase family Os08g34770 Os.38884.2.S1_x_at 4.6Os03g28930 Os.38884.2.S1_x_at 4.6Os12g35100 Os.38884.2.S1_x_at 4.6Os09g03180 Os.41695.3.S1_x_at 1.9Os02g03240 Os.41695.3.S1_x_at 1.9Os05g07920 Os.41695.3.S1_x_at 1.9Os04g23470 Os.41695.3.S1_x_at 1.9

(Table continues on following page.)





were immunoprecipitated with antibodies against acet-ylated histone H3 or acetylated histone H3K9. The pre-cipitated chromatin DNA was analyzed by real-timePCR to test for enrichment relative to nonprecipitated(input) genomic DNA. The enrichment of promoterfragments relative to input chromatin DNA in the wildtype (arbitrarily assessed as 100%) was compared tothat found for the transgenic plants. As shown in Figure8, H3K9 acetylation was induced on both the testedDNA and retroelements. H3K9 acetylation was clearlyinduced on HSR201 and APO, in agreement with theexpression data. In addition, the overall H3 acetylationwas significantly induced on the DNA elements andHSR201. This suggested an induction of acetylationof other H3 Lys residues by H3K9 acetylation in thechromatin regions, as there is an agonistic relationshipbetween different Lys residues for acetylation (Strahland Allis, 2000). The increase of acetylation on SAG12could also be observed, but to a lesser extent. No in-crease of H3K9 acetylation was observed on PR10,supporting the above-proposed hypothesis that the PRgenes might not be direct targets of OsSRT1. However,transposons and retroelements, as well as some PCDmarker genes (HSR201 and APO), might be among theprimary targets of deacetylation induced by OsSRT1.

DISCUSSION

Compared to other eukaryotes, plants have rela-tively fewer SIR2-related genes. This would suggest

that the plant members may have a larger spectrum offunctions compared to their yeast or animal counter-parts. For instance, human SIRT1, SIRT6, and SIRT7are localized to nucleus, but SIRT1 has been shown toregulate nonhistone proteins involved in apoptosis,cell survival, transcription, and metabolism. Our datashowed that OsSRT1 is nuclear localized, suggestingthat the rice protein may function mainly in the nu-cleus. Alternatively, the smaller number of SIR2-relatedgenes found in plants may be compensated by otherplant HDAC genes, as plants possess an additionalclass of HDAC genes, namely, HD2 (Lusser et al., 1997;Pandey et al., 2002). This plant-specific class of HDACslacks sequence similarity with other classes of HDACsthat are conserved from plants to humans (Pandeyet al., 2002). Arabidopsis HD2-related proteins (namedHDT1–HDT4) are nucleolus localized. Analysis of theArabidopsis family members identifies HDT1 as a geneimportant for silencing of one parental set of ribosomalRNA genes in a genetic hybrid, an epigenetic phenom-enon called nucleolar dominance (Lawrence et al., 2004).In addition, it has recently been shown that ArabidopsisHDA6, a member of the RPD3-type of HDACs, is alsoinvolved in the rDNA silencing (Earley et al., 2006).

Both of the identified plant SIR2-related genes weresignificantly divergent from yeast SIR2, as they werefound in different subclasses. Deletion of yeast SIR2caused increases of histone acetylation within therDNA region. However, we did not detect any changesof histone acetylation on rice rDNA in the OsSRT1

Table II. (Continued from previous page.)

Transposon Class Family Locus Affymetrix Probe Sets Log2

Os10g26360 Os.41695.3.S1_x_at 1.9Os03g18060 Os.41695.3.S1_x_at 1.9Os03g47130 Os.41695.3.S1_x_at 1.9Os07g46020 Os.41695.3.S1_x_at 1.9Os04g12520 Os.41695.3.S1_x_at 1.9Os05g25470 Os.41695.3.S1_x_at 1.9Os06g29050 Os.41695.3.S1_x_at 1.9Os05g36060 Os.41695.3.S1_x_at 1.9Os08g28990 Os.41695.3.S1_x_at 1.9Os06g08070 Os.41695.3.S1_x_at 1.9Os01g18470 Os.41695.3.S1_x_at 1.9Os12g29530 Os.41695.3.S1_x_at 1.9Os01g28740 Os.41695.3.S1_x_at 1.9Os07g41430 Os.41695.3.S1_x_at 1.9

MuDR transposase family Os03g46080 Os.41695.3.S1_x_at 1.9Os05g05090 Os.41695.3.S1_x_at 1.9Os11g31830 Os.41695.3.S1_x_at 1.9Os08g19970 Os.41695.3.S1_x_at 1.9Os12g08080 Os.41695.3.S1_x_at 1.9

Unclassified Os10g42160 Os.48027.1.S1_x_at 3Os09g31940 Os.52004.1.S1_at 3.3Os12g32140 OsAffx.14528.2.S1_x_at 2.2Os05g03800 OsAffx.14528.2.S1_x_at 2.2Os06g47420 Os.5212.2.S1_at 1.9Os02g43370 OsAffx.2947.1.S1_at 1.6Os12g42190 Os.19899.2.S1_at 1.6

Huang et al.




RNAi plants, in which overall rDNA expression seemednot to be affected (data not shown). Accordingly, theOsSRT1 protein seemed not to be confined or enrichedin the nucleolus (Fig. 2B). Instead, our data showedclear increases of H3K9 acetylation on the testedDNA transposable elements and retroelements in theOsSRT1 RNAi plants. The increases of H3K9 acetyla-tion were in agreement with the transcriptional acti-vation of the DNA and retroelements. In addition, ourdata showed that the down-regulation of OsSRT1 alsoaffected H3K9 acetylation and expression of hyper-sensitive response and PCD marker genes, and in-duced apoptotic cell death on leaves. In agreementwith the data, overexpression of OsSRT1 decreasedH3K9 acetylation and exhibited enhanced tolerance toan oxidative agent. OsSRT1 is found in the same class(IV) as human nucleus-localized HsSIRT6 and HsSIRT7proteins, but OsSRT1 is more closely related to HsSIRT6than HsSIRT7. HsSIRT6 has a weak in vitro HDAC ac-tivity. SIRT6 knockout mice display a deficiency inDNA repair and genomic instability (Mostoslavskyet al., 2006). In contrast, no NAD1-dependent HDACactivity is found in SIRT7 that is localized in the nu-cleolus. SIRT7 has recently been shown to promote

rDNA transcription by interacting with RNA poly-merase I (Ford et al., 2006). Therefore, it is likely thatOsSRT1 has a divergent function from SIR2-relatedproteins in yeast and mammalian cells.

We showed that OsSRT1 was widely expressed inrice, with highest levels in active cell dividing organs/tissues. Down-regulation of OsSRT1 by RNAi inducedlesion mimic cell death and precocious senescence,whereas overexpression showed tolerance to oxidativestress. These data suggest that OsSRT1 is involved inthe safeguard against genome instability and/or oxi-dative stress, required for plant cell growth. Histo-chemical staining, TUNEL assays, and molecularmarker gene analysis demonstrated that cell deathwas induced in OsSRT1 RNAi plants. The TUNELpositive signals detected in nuclei of the RNAi leafcells were indicative of DNA fragmentation, support-ing the occurrence of apoptotic PCD in the RNAiplants. However, the production of H2O2 and activa-tion of HSR201 suggested that the cell death in theOsSRT1 RNAi plants also resembled hypersensitiveresponse-mediated PCD. Either both types of PCDwere induced by OsSRT1 down-regulation, or differ-ent triggers of PCD may be interdependent in plants

Figure 7. Semiquantitative RT-PCR analysis of transposable elements (A) and PCD-related genes (B) of RNAs isolated from wildtype (WT), OsSRT1 RNAi (LM-1, -2), or overexpression (OX-1, -2) plants at days 7, 11, and 21 after germination, respectively.





and the downstream effectors of PCD may be sharedamong different pathways. In plants, it is not knownwhether PCD occurs in response to DNA damage as adefense mechanism, as DNA damage-induced PCDin mammalian cells requires the activation of p53

(Chowdhury et al., 2006) that has not been identified inplants. Therefore, it is not clear at this stage whetherthe DNA damage was the cause or the consequence ofthe PCD induced by the OsSRT1 RNAi. It is also pos-sible that DNA fragmentation was due to genome in-stability induced by oxidative stress as a result of H2O2production or transcriptional activation of transpo-sons due to increased H3K9 acetylation, a hallmark ofgene activation, as transposons can be activated in re-sponse to stress challenges.

Molecular analysis showed an increase of H3K9acetylation and a decrease of H3K9 dimethylation inOsSRT1 RNAi leaves. Since H3K9 dimethylation isfound to be mainly associated with inactive chromatin,these data suggest that OsSRT1 is needed for deacety-lation and subsequent dimethylation of H3K9 to inac-tivate chromosomal domains. The early activation oftransposable elements and two of some PCD markergenes, along with the increase of H3K9 acetylation onthese genes, suggests that both transposable elementsand protein-coding genes may be among the primarytargets of OsSRT1. Whether OsSRT1 is directly in-volved in the repression of the targets requires furtheranalysis.

The activated transposable elements were silent inwild-type plants as judged by the hybridization signals

Figure 8. Effect of OsSRT1 RNAi on histone H3 and H3K9 acetylation on transposable elements (A) and PCD-related genes (B).Nuclei were extracted from cross-linked rice seedlings, sonicated, and immunoprecipitated with antibodies specific toacetylated histone H3 (aceH3), acetylated H3K9 (aceH3K9), or without antibody (mock). The immunoprecipitates wereanalyzed by real-time PCR. The primer sets (arrowheads) are numbered for each gene, and the positions of the primers relative tothe initiation ATG codon are indicated. The relative amounts of the PCR products compared to input chromatin from wild-typeextracts (arbitrarily given as 100) are shown below the genes. Gray bars, OsSRT1 RNAi; white bars, wild type. Small barsrepresent SD from at least three repetitions.

Table III. Expression changes of apoptosis- and defense-relatedaffected genes

PCD-Related

GenesLocus Function Log2-Fold

HSR203J Os05g33940 Hypersensitive-relatedproteins

20.3

HSR201 Os12g27254 Hypersensitive-relatedproteins that areinduced duringbacterial infectionsof plant tissues

3

APO Os03g037140 Apoptosis; defense-relatedcytochrome P450

3.3

OsSAG12 Os09g32230 Senescence-specificCys protease

3.4

OsSAG13 Os03g16230 Senescence-associatedGene13

0

PR1a Os07g03690 PR protein 0.7PR5 Os12g38150 PR protein 0.6PR8 Os10g28080 PR protein 0.7PR10 Os03g18850 PR protein 2.3

Huang et al.




that were at near background levels, suggesting thesetransposable elements might be within silent hetero-chromatin domains that are known to be associatedwith DNA methylation (Tariq et al., 2003). Analysis ofgenomic DNA isolated from wild-type and OsSRT1RNAi and overexpression plants revealed two of thetested elements had decreased symmetric cytosinemethylation (as revealed by MspI and HpaII digestion)induced by OsSRT1 RNAi and two others had nochanges (Supplemental Fig. S2). However, digestionwith HaeIII, which could detect asymmetric cytosinemethylation, did not reveal clear differences betweengenome DNAs isolated from the wild-type and thetransgenic plants. Therefore, down-regulation of OsSRT1was likely to have a stochastic effect on DNA methyla-tion. This may lead to suggest that histone deacetylationmay play a primary role in OsSRT1-mediated transpo-son repression. This is in agreement with the observa-tions of Lippman et al. (2003), showing that there are twodistinct mechanisms to silence transposons in Arabi-dopsis, each of which involves different components ofchromatin modification and remodeling with histonedeacetylation as a common intermediate, as loss of func-tion mutation of HDA6 derepresses most of the testedtransposable elements. Our data with OsSRT1 suggestthat members from different classes of plant HDACgenes are involved in histone deacetylation required fortransposon silencing.

MATERIALS AND METHODS

Gene Cloning and Sequence Analysis

The cDNA fragments of OsSRT1 were amplified from rice (Oryza sativa L.

sp. indica) ‘Minghui63’ by RT-PCR. Two micrograms of total RNA from young

panicles were reverse transcribed in a total volume of 20 mL with 0.5 mg oligo

(dT)15, 0.75 mM dNTPs, 10 mM dithiothreitol, and 100 units SuperScript II

RNase H2 reverse transcriptase (Invitrogen). The following PCR primers

were designed: SRT1-F (5#-GGGGGTACCGAGAGATGTCACTTGGCTA-

TGC-3#; a KpnI site was introduced and is underlined) and SRT1-R

(5#-GGGGGATCCCCAGCTTTCACATGCACTAG-3#; a BamHI site was intro-

duced and is underlined). ExTaq DNA polymerase (TaKaRa) was used to

amplify with the following cycling profile: 94�C for 3 min; 30 cycles of 94�C for

1 min, 57�C for 1 min, and 72�C for 2 min; and extension at 72�C for 10 min.

The PCR product was cloned into the pGEM-T vector to obtain the pT237

clone (Promega) and confirmed by sequencing from both ends.

For sequence analysis, all of the SRT family sequences that were used for

sequence alignment and phylogenetic analysis were downloaded from the

plant ChromDB database (http://www.chromdb.org/). The sequences of the

Sir2 domain (Pfam accession no. PF02146) were searched in the Pfam database

(http://pfam.janelia.org/). Phylogeny reconstruction of all Sir2 domain se-

quence alignments was performed by MEGA 3.1 (Kumar et al., 2004) using the

neighbor-joining method with a Poisson correction model and a bootstrap of

500 replicates.

Nuclear Localization

The nuclear localization vector was constructed by replacing the GUS frag-

ment of pCAMBIA1391Xb (CAMBIA) with a ubiquitin promoter-GFP cas-

sette. The coding region of OsSRT1 cDNA was amplified using the following

primer pair: FU-F 5#-GGGGAATTCTCGGGAGAAGCTTACTTGATTGAG-3#(an EcoRI site was introduced and is underlined) and FU-R 5#-GGGGGATCC-

ACTGATCGAAGAAATGGCAAAGG-3# a BamHI site was introduced and is

underlined). The amplified fragment was inserted upstream to and in frame

with GFP. The procedure of bombarding onion (Allium cepa) epidermal cells

was as described (Dai et al., 2007). The expression of the fusion protein of

OsSRT1 and GFP in the onion epidermal cells was observed by a confocal

microscope (Leica) 36 h after bombardment.

Vector Construction and Rice Transformation

A 412-bp cDNA fragment of OsSRT1 was amplified using primers RNAi-F

(5#-GGGACTAGTGGTACCAGTCCTGCAAGAGTTGCAAC-3# with a SpeI site,

bold letters, and a KpnI site, underlined integrated) and RNAi-R (5#-GGG-

GAGCTCGGATCCCCAGCTTTCACATGCACTAG-3# with a SacI site and a

BamHI site). PCR products were digested with KpnI/BamHI and SacI/SpeI,

respectively, and inserted into pDS1301 (Chu et al., 2006).

The overexpression vector was constructed by directionally inserting the

full cDNA sequence (digested with BamHI/KpnI) into the binary vector pU1301,

which was modified based on pCAMIA1301 (CAMBIA) and contained a maize

(Zea mays) ubiquitin promoter. Agrobacterium tumefaciens (strain EHA105)-

mediated transformation of rice plants was conducted according to a published

protocol (Lin and Zhang, 2005).

Expression Analysis by Northern Blots, Microarray,

and RT-PCR

For northern-blotting analysis, 15 mg of total RNA samples extracted from

tissues or organs harvested from field-grown rice plants was separated in 1.2%

(w/v) formamide-denaturing agarose gels, before being transferred to nylon

membranes. Gene-specific probes were labeled with 32P-dCTP using the

Random Primer kit (Invitrogen) and hybridized to the RNA blots. The probe

of OsSRT1 was digested from pT237 plasmid with KpnI and SpeI, a fragment of

635 bp of the cDNA.

For microarray analysis, transgenic and wild-type seedlings were grown in

half-strength Murashige and Skoog medium under a 16-h-light/8-h-dark

cycle at 25�C for 11 d, for three biological repeats. RNA samples were ex-

tracted using TRIzol (Invitrogen) as described by the manufacturer. Hybrid-

ization with Affymetrix GeneChip Rice Genome Arrays was performed at

CapitalBio Corporation. The dataset was normalized with the option of all

probe sets scaled to the target signal of 100. The genes with expression calls as

absent from at least 11 arrays were filtered, resulting in 17,806 genes for

further analyses. The significance analysis of microarrays (SAM) Excel add-in

(Tusher et al., 2001) was used to identify significantly differentially expressed

genes between the control and RNAi seedlings. The imputation engine was set

as 10-nearest neighbor imputer and the number of permutations was 100. The

delta value in the SAM was adjusted so that the estimated false discovery rate

was ,5% for significant genes.

For semiquantitative RT-PCR analysis, 2 mg of total RNA was reverse-

transcribed in a total volume of 20 mL with 0.5 mg oligo(dT)15, 0.75 mM dNTPs,

10 mM dithiothreitol, and 100 units of SuperScript II RNase H2 reverse

transcriptase (Invitrogen). PCR was performed in a total volume of 20 mL with

1 mL of the RT reactions, 0.2 mM gene-specific primers, and 1 unit of rTaq

(TaKaRa). Twenty-five to 30 cycles were performed. Rice actin cDNA was used

as internal control. The sequences of the used primers are listed Supplemental

Table S1.

DAB Assays

The DAB uptake method (Thordal-Christensen et al., 1997) was used to

detect H2O2. Ten-day-old seedlings were placed in DAB dissolved in 10 mM

ascorbic acid (1 mg/mL) for 2 h. The seedlings were then boiled in 96%

ethanol for 10 min and stored in 96% ethanol. H2O2 production was visualized

as reddish-brown coloration.

TUNEL Assays

The TUNEL assays were performed using the In Situ Cell Death Detection

Kit-Fluorescein (Roche Diagnostics). Leaf tissues from wild-type and RNAi

plants were fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline

(pH 7.2) containing 0.1% (v/v) Triton X-100 and Tween 20 at 4�C overnight

and embedded in paraplasts. Eight-micrometer sections on glass slides were

dewaxed in xylene, rehydrated, and then pretreated with 20 mg/mL protein-

ase K in 10 mM Tris-Cl, pH 7.5, for 20 min at room temperature. Two slides,

treated with 1,500 units/mL DNase I in 50 mM Tris-Cl, pH 7.5, 1 mM MgSO4,

and 1 mg/mL bovine serum albumin for 20 min at room temperature, served

as positive controls. Two slides, labeled in the absence of the terminal deoxy-

ribonucleotidyl transferase enzyme, served as negative controls. Vectashield





mounting medium (Vector Laboratories) and protease inhibitors (1 mg/mL;

Sigma) were used to mount the slides before they were viewed and photo-

graphed with a Leica microscope.

Paraquat Treatment

For paraquat treatments, leaves of 14-d-old plants (three replications,

50 per treatment) were exposed to a surface application of 10 mM paraquat

(Sigma) in a 0.1% solution of the nonionic surfactant Ortho X77 (Valent USA)

5 h during the light period. The controls were treated with 0.1% surfactant

alone (Donahue et al., 1997).

Western-Blot Analysis

Rice leaf histone protein extraction was performed as described (Tariq

et al., 2003). After washing in acetone and dried, the proteins were resuspended

in Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% glycerol,

0.01% bromophenol blue, and 10% b-mercaptoethanol), then separated on a 16%

SDS-PAGE and transferred to an Immobilon-P PVDF transfer membrane

(Millipore). The membrane was blocked with 2% bovine serum albumin in

phosphate-buffered saline (pH 7.5), and incubated overnight with primary

antibodies, such as anti-acetylated histone H3, anti-dimethyl-histone H3K9,

and anti-acetyl-histone H3K9 (from Upstates; catalog nos. 06–599, 07–441,

and 07–352, respectively) in a 1:5,000 dilution at room temperature. After

three washes (30 min each), the secondary antibody (goat anti-rabbit IgG

[SouthernBiotech]) at 1:10,000 dilution was used. Visualization was performed

using the SuperSignal West Pico kit (Pierce) according to the manufacturer’s

instructions.

ChIP Assay

ChIP assays were performed as described (Benhamed et al., 2006).

Immunoprecipitated DNA was analyzed by real-time PCR (Applied Biosys-

tems 7500). Primers were designed by PRIMER EXPRESS 2.0 software (PE

Applied Biosystems) to amplify 80- to 120-bp products. Products were

measured by SYBR green fluorescence (Applied Biosystems) in 25-mL reac-

tions; all primers were annealed at 58�C. Data analyses with 22DDCt method

were performed as described (Livak and Schmittgen, 2001). Real-time PCR

primers are listed in Supplemental Table S2.

DNA Methylation Analysis

Genomic DNA (1 mg) was digested for 6 h at 37�C with 30 units of HpaII,

MspI, or HaeIII. Five percent of the digested and input (undigested) DNA was

analyzed by PCR (Onodera et al., 2005). PCR conditions were as follows: 5 min

at 96�C, followed by 35 cycles of 94�C for 1 min, 58�C for 1 min, and 72�C for

1 min. Primers used for PCR are listed in Supplemental Table S3.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number XP_471492.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Transgenic siblings without OsSRT1 overex-

pression showed no enhanced tolerance to paraquat.

Supplemental Figure S2. Analysis of cytosine methylation of transposons

in OsSRT1 overexpression or RNAi plants.

Supplemental Table S1. Primers used for RT-PCR analysis.

Supplemental Table S2. Primers used for real-time PCR analysis.

Supplemental Table S3. Primers used for DNA methylation analysis.

ACKNOWLEDGMENTS

We thank Li Xianhua for technical assistance, Xu Caiguo for rice field

management, and Song Huazhi for assistance with confocal microscopy. We

acknowledge Qiu Deyun and Xie Weibo for help in microarray data analysis.

Received March 13, 2007; accepted April 17, 2007; published April 27, 2007.

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