Arabidopsis SHORT INTEGUMENTS 2 Is a Mitochondrial DAR …

Copyright � 2006 by the Genetics Society of AmericaDOI: 10.1534/genetics.106.060657

Arabidopsis SHORT INTEGUMENTS 2 Is a Mitochondrial DAR GTPase

Theresa A. Hill,1 Jean Broadhvest,2 Robert K. Kuzoff3 and Charles S. Gasser4

Section of Molecular and Cellular Biology, University of California, Davis, California 95616

Manuscript received May 16, 2006Accepted for publication July 12, 2006

ABSTRACT

The Arabidopsis short integuments 2-1 (sin2-1) mutant produces ovules with short integuments due toearly cessation of cell division in these structures. SIN2 was isolated and encodes a putative GTPase sharingfeatures found in the novel DAR GTPase family. DAR proteins share a signature DAR motif and a uniquearrangement of the four conserved GTPase G motifs. We found that DAR GTPases are present in allexamined prokaryotes and eukaryotes and that they have diversified into four paralogous lineages inhigher eukaryotes. Eukaryotic members of the SIN2 clade of DAR GTPases have been found to localize tomitochondria and are related to eubacterial proteins that facilitate essential steps in biogenesis of thelarge ribosomal subunit. We propose a similar role for SIN2 in mitochondria. A sin2 insertional allele hasovule effects similar to sin2-1, but more pronounced pleiotropic effects on vegetative and floral devel-opment. The diverse developmental effects of the mitochondrial SIN2 GTPase support a mitochondrialrole in the regulation of multiple developmental pathways.

ARABIDOPSIS ovules are a useful model system forstudying developmental mechanisms. Arabidopsis

ovules initiate on the inner surface of immature carpelsas relatively featureless finger-like primordia. As theyelongate, three different regions become specializedand undergo morphogenesis and cellular differentia-tion (Robinson-Beers et al. 1992). The distal-most re-gion, the nucellus, is the site of meiosis and embryosac formation. The central chalazal region is the site ofthe most visible morphogenic changes as it gives riseto two appendages, the inner and outer integuments.The basal region elongates through division and coor-dinated expansion of cells forming the funiculus, asupporting stalk. During this process, the developingovule becomes bilaterally symmetrical as a result ofdifferential growth that causes the funiculus to curvetoward the base of the carpel and the outer integumentto curve toward the carpel apex. At maturity, bothinteguments have grown to enclose the nucellus andform a terminal micropylar opening (Figure 1A). Mu-tations altering ovule morphogenesis may also disruptother plant developmental pathways and the relative

morphological simplicity of ovules can facilitate over-all understanding of the underlying biochemical ormolecular processes.

Numerous genes affecting growth and patterning ofovules have been identified via mutagenesis and cloning(Schneitz 1999; Skinner et al. 2004). Several of thegenes regulating Arabidopsis ovule development man-ifest their effects through alterations in the pattern orprogress of cell division. These genes encode proteinswith a variety of biochemical functions (Skinner et al.2004). For example, mutations in AINTEGUMENTA(ANT) and INNER NO OUTER (INO), encoding AP2and YABBY-domain transcription factors, respectively,result in a complete absence of both integuments or ofonly the outer integument (Gaiser et al. 1995; Elliott

et al. 1996; Klucher et al. 1996; Baker et al. 1997;Villanueva et al. 1999). TSO1 encodes a novel nuclearprotein required for proper orientation of cell elonga-tion and cytokinesis during floral organ and integumentdevelopment (Hauser et al. 1998, 2000; Song et al.2000). Severe mutations in the HUELLENLOS (HLL)gene, encoding a mitochondrial L14 ribosomal subunit,lead to an arrest in ovule growth and degeneration ofthe apical regions of the primordia, revealing a role formitochondrial activity in the process of ovule growth(Schneitz et al. 1998; Skinner et al. 2001). Among otherfloral effects, reduced activity of the putative proteinkinase TOUSLED (TSL) causes short outer and pro-truding inner integuments (Roe et al. 1997a,b, 1993).The variety of protein classes involved in ovule growthimplies complex regulation of this process at the levelsof transcription, signal transduction, and metabolism.

SHORT INTEGUMENTS 2 (SIN2) is required for sus-taining cell divisions during integument development

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. AY254472.

1Present address: USDA ARS WRRC-GGD, 800 Buchanan St., Albany,CA 94710.

2Present address: Bayer BioScience N.V., Technologiepark 38, 9052Ghent, Belgium.

3Present address: Department of Biological Sciences, University ofWisconsin, Whitewater, WI 53190.

4Corresponding author: Section of Molecular and Cellular Biology,University of California, 1 Shields Ave., Davis, CA 95616.E-mail: [email protected]

Genetics 174: 707–718 (October 2006)

(Broadhvest et al. 2000). At anthesis, sin2-1 ovules havefewer integument cells than wild type, leaving theirnucelli exposed (Figure 1B). sin2-1 also has subtlepleiotropic effects on flower development. The gynoe-cia of sin2-1 mutants generally have a cleft stigma andoccasionally bear an outgrowth on the correspondingvalve. Additionally, in sin2-1 sepals marginal cells ap-pear to be absent or highly reduced in number. Thesepleiotropic effects suggest that SIN2 facilitates severalmorphogenic pathways.

Double mutants with sin2-1 revealed functional re-lationships between SIN2 and other ovule develop-ment genes (Broadhvest et al. 2000). ant-5 sin2-1double mutants were indistinguishable from ant-5 mu-tants, but ant and sin2 had similar synergistic inter-actions with hll. Both ant hll and sin2-1 hll doublemutants exhibited a reduced number of ovules andan earlier abortion of primordia development thanhll single mutants. The ovule effects of sin2-1 wereadditive with the cell expansion defects of tso1-3. ThusSIN2 may act downstream of ANT in a common pathwaywith HLL, and in parallel to TSO1, to promote cell

division during integument growth (Broadhvest et al.2000).

To better understand regulation of cell divisionduring organ formation, we have identified and char-acterized the SIN2 gene and isolated a second allele,sin2-2. SIN2 encodes a putative GTPase of a relativelyuncharacterized subclass, termed the DAR GTPases,on the basis of a conserved aspartate–alanine–arginine(DAR) motif and other conserved features (Fu et al.1998). Some DAR GTPases have been found to be im-portant for cell division in bacteria, fungi, and humanstem cell lines, where they are associated with assemblyor subcellular transport of ribosomal subunits (Bassler

et al. 2001; Saveanu et al. 2001; Bialkowska andKurlandzka 2002; Morimoto et al. 2002; Tsai andMcKay 2002; Kallstrom et al. 2003; Matsuo et al.2006; Uicker et al. 2006). We found SIN2 to localize tomitochondria and hypothesize a function in mitochon-drial ribosome assembly. In conjunction with hll, sin2mutants provide an attractive system with which to studythe role of mitochondrial function in the developmentof a multicellular organism.

Figure 1.—Scanning electron micrographs ofstage 3-VI ovules (anthesis; stages from Schneitz

et al. 1995). (A) Wild-type Ler. (B) sin2-1. (C andD) sin2-2 plants. In D, the gradation in severity ofeffects of sin2-2 from the base to the apex of thecarpel is apparent. f, funiculus; ii, inner integu-ment; n, nucellus; oi, outer integument. Bar,50 mm (A–C) and 100 mm (D).

708 T. A. Hill et al.

MATERIALS AND METHODS

Plant material: Plants were grown on soil as previouslydescribed (Kranz and Kirchheim 1987; Robinson-Beers

et al. 1992). Some plants were germinated on 1% agar con-taining 1% sucrose, 13 Murashige and Skoog (MS) salts, and13 Gamborg’s B-5 vitamins (Murashige and Skoog 1962;Gamborg et al. 1968). The Student’s t-test was used to de-termine if mutant plants were significantly different fromwild-type plants in germination rate, number of days toflowering, number of rosette leaves produced, leaf produc-tion rates, and number of axillary meristem elongations.Plant transformation vectors and methods were as described(Meister et al. 2002).

Genetic mapping and complementation: Using a mappingpopulation generated by crossing sin2-1 (Landsberg erecta,Ler) with wild-type Columbia (Col-3), SIN2 was previouslylocalized to chromosome 2 between the cleaved amplifiedpolymorphic sequence (CAPS) m429 and the simple sequencelength polymorphism (SSLP) AthBIO2 (Broadhvest et al.2000). Of 31 chromosomes that had a recombination eventbetween m429 and AthBIO2, four recombination points werebetween SIN2 and the SSLP marker F13H10 Indel2 [Cereonaccession CER448978; The Arabidopsis Information Resource(TAIR) http://www.arabidopsis.org/Cereon/] and one recom-bination was between SIN2 and a ClaI RFLP within T11A7.16(At2G41740). This region is spanned by three overlappingbacterial artificial chromosome (BAC) clones (T11A7, T1K18,and T32G6; Choi et al. 1995). Cosmid subclones were gen-erated from BAC T1K18 by partial digestion with Sau3AI andinsertion of 12- to 25-kb fragments in pOCA28-15 (Eshed et al.1999). The overlapping cosmids cJBT1K18.136, cJBT1K18.105,and CJBT1K18.107 were identified and assembled into acontig using hybridization and fingerprint data. These cos-mids spanned the annotated genes At2G41600.1–At2G41730.1and were transformed into plants from the sin2-1 mappingpopulation. The genotype of transgenic plants at the SIN2locus was determined with the F13H10 Indel2 SSLP and theDdeI CAPS marker 6D20R in At2G42090.1.

Molecular methods: Coding regions for genes within thecomplementing cosmid (cJBT1K18.136) were isolated fromsin2-1 homozygous and Ler plants by reverse transcriptionpolymerase chain reaction (RT–PCR) using gene-specificprimers and SuperscriptII reverse transcriptase (Invitrogen,Carlsbad, CA). The cDNAs were sequenced by Davis Sequenc-ing (Davis, CA). Sequences were compared using Sequencher(Gene Codes, Ann Arbor, MI). Genomic subclones, includingeach gene within the complementing cosmid along with thesequences 59 and 39 extending into the next closest genes,were isolated from cJBT1K18.136 and transformed into plantsfrom the sin2-1 mapping population. For At2g41670 (SIN2),a 4.0-kb EcoRI fragment was isolated and inserted into thesesame sites in pBJ97 (Gleave 1992), creating pTH13. At2g42670was removed from pTH13 on a NotI fragment and insertedinto the same site in pMLBART (Gleave 1992) for plant trans-formation. The 59-end of the SIN2 cDNA was determined bysequencing clones obtained with the GeneRacer 59 RACESystem (Invitrogen) using primers specific to SIN2. The full-length cDNA was inserted as an SpeI/BamHI fragment intoXbaI/BamHI-digested pMON999 (Meister et al. 2002), creat-ing a P35STSIN2TNOS39 transcriptional fusion (pTH52).The cDNA was also attached to a 5.6-kb SIN2 59-flankingregion using a common NruI site in the first exon to createpTH64. The gene fusions in pTH52 and pTH64 were trans-ferred to pMLBART (Gleave 1992) on NotI fragments forplant transformation.

Primers designed to amplify the SIN2 genomic region,SIN2KOF2 (GAT GGG TTA TTA CGA TTT GGG CAG TTA

TT) and SIN2KOR (CAG TTT CAG GGA CAT CGT CAA GGATAA AG), were used to screen the Wisconsin a-population ofinsertion lines for additional sin2 alleles as described byKrysan et al. (1999). The insertion site in sin2-2 was deter-mined by sequencing PCR products using SIN2 primers oneither side of the insertion and the T-DNA JL202 primer. TheSIN2KOR and JL202 primers (Krysan et al. 1999) were used todetect the sin2-2 insertion in segregating populations.

An RT–PCR product generated using synthetic primersT32G6.19F and SIN2GR on first-strand cDNA from Ler in-florescence poly(A)1 RNA was cloned into the pCR2.1 vector(Invitrogen), forming pTH19. The insert was sequenced andfound to include the entire SIN2 coding region with nomutations except for those intentionally introduced to removethe stop codon. This cDNA was mobilized on an SpeI/BamHIfragment and inserted into pRJM86 (Meister et al. 2002) atthe same sites to form a fusion with the green fluorescentprotein (GFP) 1.1.15 (Schumacher et al. 1999) coding region.The SpeI/KpnI fragment containing the SIN2:GFP fusionprotein-coding region was inserted between the CaMV 35Spromoter and the nopaline synthase polyadenlyation region(NOS39) in pMON999 (Meister et al. 2002). A NotI fragmentconsisting of P35STSIN2:GFP NOS39 was inserted into theNotI site of the plant transformation vector pMLBART(Gleave 1992) and transformed into plants.

Microscopy: Samples were prepared for scanning electronmicroscopy and images were acquired and edited as described(Broadhvest et al. 2000). Samples were fixed for lightmicroscopy as described by Baum and Rost (1996). Forviewing petal veins, following fixation, flowers were clearedovernight in a saturated aqueous chlorylhydrate solution.Petals were removed from the flowers and observed with aZeiss Axioplan (Zeiss, Oberkochen, Germany) microscopeusing dark-field optics. GFP localization was determined usingtransgenic SIN2:GFP T2 seedlings, which were stained for 1 hrin a 13 MS solution containing the mitochondrial-specificstain MitoFluor Red589 (Molecular Probes, Eugene, OR) at500 nm and were visualized on a Zeiss Axioscope microscopeusing fluorescein isothiocyanate and tetramethylrhodamineisothiocyanate filter sets, respectively. Images were recordedusing the Openlab system (Improvision, Lexington, MA) andwere adjusted for contrast in Adobe Photoshop 6.0 (AdobeSystems, San Jose, CA).

Phylogenetic analysis: BLAST (Altschul et al. 1997)searches were used to identify sequences in GenBank encod-ing proteins similar to SIN2 and other Arabidopsis DARproteins. A large number of proteins containing G motifswere identified. DAR proteins (defined as those including aDAR-G4-G1-G2-G3 arrangement of motifs but which did nothave two complete G domains in tandem; Fu et al. 1998)uniformly gave the best matches, followed by ENGA proteins.Parsimony analysis was performed with PAUP4.0b6 (Swofford

1999) on alignments created using ClustalX (Thompson et al.1997). Three separate alignments were produced using gapopening:substitution costs of 2.5:0.5, 5:0.4, and 10:0.3, and theBLOSUM series of weightings (Henikoff and Henikoff

1992). ENGA sequences from the ENGA DAR motif to thesecond G3 motif were used as an outgroup. Regions unstablebetween alignments were detected using SOAP (Loytynoja

and Milinkovitch 2001). Sequences ,70% stable were notincluded in the phylogenetic analysis (culling). A secondanalysis was performed in which all three alignments, ar-ranged in tandem, were included (elision; Wheeler et al.1995). In parsimony analysis, characters were weighted using aBLOSUM45 amino acid substitution matrix, which was gener-ated from the published BLOSUM45 matrix (Henikoff andHenikoff 1992) by substituting ‘‘15 � n’’ for each value ‘‘n’’of the matrix. This results in all positive values, ranging from

SHORT INTEGUMENTS 2 Is a Mitochondrial GTPase 709

0 to 20. An amino-acid-to-gap transition was given a cost equalto the highest value in the matrix, 20. Bootstrap values werecalculated from 500 bootstrap replicates of heuristic searcheson 10 random sequence additions per bootstrap replicate andtree bisection–reconnection procedure for branch swapping.Branches that were not consistent between the culling andelision methods of character weighting or those that hadbootstrap values #50% were collapsed.

Accession numbers: The GenBank/EMBL accession num-ber for the SIN2 cDNA is AY254472. The accession numbersfor other protein sequences used in the analysis are: Arabi-dopsis DGP2 (CAB40031); Arabidopsis DGP3 (AAD15335);Arabidopsis DGP4 (AAF27009); Arabidopsis DGP5 (AAG52287);Arabidopsis DGP6 (AAD26884); Arabidopsis DGP7 (AAF22888);Oryza DGP4 (BAB61154); Oryza DGP3 (AU077558); OryzaDGP1 (AAAA01011536); Oryza DGP2 (AC077693); MedicagoDGP6 (BG452200); Hordeum DGP5 (BF619769); Zea DGP6(AAD41267); Lycopersicon DGP1 (BG133077); LeishmaniaDGP3 (CAC32254); Leishmania DGP2 (CAC32261); Leish-mania DGP1 (AAF73086); Bacillus ylqF (F69880); BorreliaDGP (AAC67000); Vibrio DGP (AAF96431); Streptococ-cus DGP (AAK75264); Synechocystis DGP (NP_441269); NostocDGP (NP_484788); Methanococcus DGP (G64482); Pyrococ-cus DGP (F75050); Pyrobaculum DGP (NP_558827); humanMTG1 (AAH04409); human LSG1 (NP_060855); humannucleostemin (GNL3, NP_055181); human GNL2 (NGP1,AAH00107); human HSR1 (XP_041722); human GNL3L(NP_061940); Drosophila DGP2 (Dme1_CG3983, AAF55384);Drosophila DGP1 (Dmel_CG6501; AAF56060); DrosophilaDGP4 (Dme1_CG14788, AAF45628); Drosophila DGP5(Dme1_CG9320, AAF53920); Drosophila Ngp (AAF57834);Caenorhabditis DGP4 (C53H9.2, AAK68267); CaenorhabditisDGP3 (T24970); Caenorhabditis nst-1(CAA88860); Caeno-rhabditis DGP1 (Y67D2.4, NM_065016); Saccharomyces LSG1(NP_011416); Saccharomyces MTG1 (S55083); Saccharomy-ces NUG1 (S50464); Saccharomyces NOG2 (NP_014451);Treponema ENGA (P96128); Aquifex ENGA (O67749);Borrelia ENGA (O51461); Rickettsia ENGA (NP_360667)

RESULTS

SIN2 encodes a putative GTPase: The ethyl-methane-sulfonate-induced sin2-1 allele was previously mappedto chromosome 2 using a sin2-1 Ler/Col segregat-ing population (Broadhvest et al. 2000). Further anal-ysis of this population localized SIN2 to a regionspanned by three overlapping clones (T11A7, T1K18,and T32G6; Choi et al. 1995). Figure 2A illustratesthe overlapping cosmid subclones of T1K18 that weretransformed into sin2-1 heterozygous progeny fromthe mapping population. Eight of the nine identifiedhomozygous sin2-1 plants (genotyped with mappingmarkers flanking the SIN2 locus) carrying cJBT1K18.136exhibited a wild-type ovule phenotype, indicating com-plementation of the mutation. cJBT1K18.136 comprisesfour complete predicted coding regions (At2g41660,41670, 41680, and 41690; TAIR, http://www.arabidopsis.org/). Subclones spanning these coding regions fromLer and sin2-1 plants were sequenced and a singlegene, At2g41670, was found to contain a C-to-T muta-tion unique to sin2-1 plants. A 4.0-kb EcoRI fragmentspanning At2g41670 (Figure 2B) was also able to com-

plement the sin2-1 mutation, confirming this gene asSIN2.

RT–PCR generated a 1.25-kb SIN2 cDNA that derivedfrom the eight exons spanning 2 kb of genomicsequence predicted for At2g41670 and encoded a 386-amino-acid protein (Figure 2B). The 59-end of the SIN2mRNA was determined by 59-RACE PCR to be 34 basesupstream of the putative translation start codon. Thecomplete coding region of the cDNA was fused to 5.6 kbof genomic sequence 59 to the SIN2 coding region or tothe CaMV35S promoter, and both constructs comple-mented the sin2-1 mutation. While we were unable todetect SIN2 transcript by in situ hybridization, transcriptwas detected by RT–PCR in all structures assayed (Figure

Figure 2.—Identification and expression of the SIN2 gene.(A) The chromosomal region surrounding the SIN2 locuswith the molecular markers used to map SIN2 shown above,and the number of recombination events between markersfound in 918 F2 plants indicated below the top horizontalline. The BAC T1K18 spans part of the region between theclosest flanking markers. Cosmid subclones .105, .136, and.107, derived from T1K18 and used in the complementationtest, are illustrated. (B) The 4.0-kb EcoRI fragment spanning asingle gene, At2G41670, derived from cJBT1K18.136, whichwas able to complement the sin2-1 mutant phenotype. Theexons in this region and the transcriptional start site as deter-mined by RT–PCR are shown as boxes and a bent arrow, re-spectively. Arrows indicate the positions of primers used toscreen T-DNA lines for additional sin2 alleles. The positionsof the missense (sin2-1) and insertional (sin2-2) mutationsare indicated. Numbering is relative to the translational startsite. (C) RT–PCR with total RNA from several plant parts usedas template shows that SIN2 RNA was present in all structuresassayed.


2C). Fusions of either the 5.6- or the 1.5-kb region 59 ofthe SIN2 coding sequence to the GUS coding regionfailed to produce GUS activity sufficient to be detectedby histochemical staining in transgenic plants. Theseobservations indicate that SIN2 is likely expressed at lowlevels in a variety of plant organs.

The predicted SIN2 protein contains the four se-quence motifs (G1–G4) typical of all members of theGTPase superfamily (Bourne et al. 1991). The sin2-1lesion produces a change of a conserved serine tophenylalanine at position 150 within the P-loop orputative GTP binding pocket present in the G1 motif(Figure 2B). Because sin2-1 was the only mutant allele ofthis gene that we had identified, and since mutations inthe G1 motif can have a gain-of-function effect (Bourne

et al. 1991), it was not possible to assess if the effects ofsin2-1 on plant development were typical of SIN2 loss offunction. Determination of the SIN2 sequence enabledscreening for an insertional allele. A transgenic line inwhich a T-DNA had inserted in the seventh exon ofSIN2, and associated with deletion of the SIN2 nucleo-tides corresponding to amino acids 281–290 at theinsertion site, was identified in the Wisconsin Knock-Out population (Krysan et al. 1999). This lesion wasdesignated sin2-2 (Figure 2B).

Plants heterozygous for sin2-2 were not visibly differ-ent from wild-type siblings, indicating that sin2-2 is arecessive allele. sin2-2 plants produced ovules similar tothose of sin2-1 mutants with a reduced number of ovuleintegument cells (Figure 1, B and C; Broadhvest et al.2000). However, the extent of sin2-2 integument re-duction was more variable, with some sin2-2 pistilsincluding as many as six ovules with a nearly wild-typeappearance in addition to those with short integuments(Figure 1D). These morphologically wild-type ovuleswere usually at the distal end of the carpel. Even with thisvariation in ovule phenotype, sin2-2 plants were com-pletely female sterile but male fertile as were sin2-1plants. The similarity of the most severe integumentphenotype between the sin2-2 insertional mutant andthe sin2-1 point mutant suggests that sin2-1 is a loss-of-function allele and that SIN2 acts to promote celldivisions in the developing integuments.

sin2-2 illuminates additional developmental roles forSIN2: sin2-1 and sin2-2 plants displayed both overlap-ping and allele-specific phenotypes. However, it ispossible that some of the specific phenotypic effectsobserved in sin2-1 and sin2-2 plants were linked to theirrespective genetic backgrounds [Ler and Wassilewskija(Ws), respectively]. Mutant phenotypes were always com-pared to those of wild-type siblings found in the samesegregating population (unless specified otherwise). Areduction in the expected 1:3 (sin2-1:wild type) segre-gation ratio had been previously noted and was pro-posed to be due to reduced viability of sin2-1 plants(Broadhvest et al. 2000). A greater degree of reductionwas observed in sin2-2 plants. When seeds from sin2-2

heterozygote plants were sown in soil, 15% failed to ger-minate, an additional 12% failed to survive to maturity,and sin2-2 mutants were underrepresented in the popu-lation of plants reaching maturity (3 of 143 total vs. 35 of143 that would be expected). However, the sin2-2 allelewas found at the expected frequency among 70 phe-notypically wild-type plants (47 of 140 chromosomes, a1:2.04 ratio of homozygotes to heterozygotes), consis-tent with Mendelian transmission of the allele. On media,the sin2-2 germination defect was partially rescued,with an overall germination efficiency of 98.5% (336 of341) in a segregating population. The underrepresen-tation of sin2-2 mutants when grown in soil indicatesthat sin2-2 plants exhibit a greater reduction in viabilityin soil than sin2-1 plants.

Vegetative growth rates were also affected in sin2mutants. Both sin2-1 and sin2-2 mutants exhibited anextended germination period. In a sin2-2 segregatingpopulation at day 13 of growth on media, 82 seedlingshad only cotyledons while 257 had developed visiblerosette leaves. Forty-eight of these seedlings with twoleaves .1 mm and 48 bearing only cotyledons weretransplanted to soil. All seedlings without leaves thatsubsequently matured to flowering were shown to behomozygous for the sin2-2 allele, demonstrating aslower germination rate for sin2-2 seeds compared towild-type siblings. Detailed analyses showed that bothsin2-1 and sin2-2 seedlings exhibited slower emergenceof radicle, cotyledons, and secondary leaves than theirwild-type siblings, with sin2-2 showing the strongesteffects in regard to germination and seedling growth(Table 1). The leaf emergence rate of sin2-2 was re-duced by half, but sin2-1 plants behaved as their wild-type siblings (Table 1). The sin2-2 plants also appeareddarker green than their wild-type siblings (supplementalFigure 1A at http://www.genetics.org/supplemental/).These alterations in germination, leaf production,and greening suggest the involvement of SIN2 activityin a variety of growth processes during vegetativedevelopment.

In addition to the reduction in integument growth,sin2 mutations affected other aspects of reproductivedevelopment. The number of rosette leaves producedprior to flowering was increased in both mutants relativeto wild type when grown in continuous light (Table 1).This alteration of flowering time appeared specific tolong-day conditions since sin2-1 mutants grown undershort-day (12 hr light) conditions produced a wild-typenumber of rosette leaves (12.63 6 1.39 vs. 12.85 6 0.96).sin2-2 mutants also showed reduced apical dominance,characterized by a doubling in the number of secondaryinflorescences, while sin2-1 did not show any obviousalterations in apical dominance (Table 1). In somecases, sin2-1 plants had multiple branches originatingfrom the axil of a single cauline leaf, resulting in a bushyphenotype (supplemental Figure 1 at http://www.genetics.org/supplemental/). sin2-2 plants also exhibited a


marked increase in vascular discontinuity within thepetals relative to wild type, sometimes exhibiting acompletely isolated vein region (supplemental Figures1 and 2 at http://www.genetics.org/supplemental/).Taken together, the pleiotropic effects of both sin2mutant alleles suggest that SIN2 may play a role in theproduction, perception, or response to a variety ofdevelopmental signals.

SIN2 is a member of a family of atypical GTPases:Database searches indicated that SIN2 shares severalconserved features, including the unique conservedaspartate–alanine–arginine (DAR) motif, with a rela-tively uncharacterized family of GTPases, the DARGTPases (Fu et al. 1998). We performed similaritysearches of all publicly available sequence databasesand identified DAR proteins from bacteria, archaea,yeast, plants, and animals. Sequence alignments gener-ated from 61 representatives of the identified proteinsrevealed additional unreported conserved features.Figure 3 shows that sequence and order conservationwas present within the four G motifs [in the non-canonical order: G4-G1-G2-G3 (Reynaud et al. 2005)],the DAR motif, and a previously unreported C-terminalmotif. In addition to its unique position, the G4 motifshowed the greatest intersequence variability among theG motifs. However, the typical G4 motif found in allGTPase superfamily proteins, (Z)4NKXD (where Z is anynonpolar or hydrophobic amino acid residue and X isany amino acid residue), was found in 80% of these DARproteins, and 7 of the 10 deviations from this consensusconsisted of conservative lysine-to-arginine or aspartate-to-glutamate substitutions. The DAR proteins were alsofound to invariably have a lysine or arginine one to threeamino acids preceding the four hydrophobic or non-polar amino acids of the G4 motif. Examination of theDAR motif region revealed two additional conservedamino acid residues, aspartate (D) and proline (P),flanking the DAR sequence, and several positions gen-erally consisting of nonpolar or hydrophobic residues,expanding the DAR consensus to D(Z)3XZXDARXP.The DAR motif was always found N-terminal to the fourG motifs in our sample. C-terminal to the G motifs, aloosely conserved sixth motif, L(X)5G, was also identi-fied. This additional motif, as well as the DAR motif,is likely to be involved in specific protein–proteininteractions.

While each prokaryotic genome examined includedonly a single DAR gene, multiple DAR genes wereobserved in eukaryotes, with seven members found inArabidopsis [SIN2 and DAR GTPases 2–7 (DGP2–7)].Maximum-parsimony analysis was used to evaluate therelationships among 51 of the identified DAR proteinsselected to represent the breadth of the entire class. Theresulting tree indicated that eukaryotic and eubacterialDAR proteins fall into four distinct clades, which wehave designated 1–4 in Figure 4A. Each clade was foundto include representatives from all major groups of

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multicellular crown eukaryotes: plants, animals, andfungi. Each clade also included at least one ArabidopsisDGP. This analysis was not sufficient to resolve therelationships between the archeal proteins and the restof the DAR proteins or among clades 1–4.

All eubacterial DAR proteins and SIN2 were includedin clade 1. Also included in this clade were the Arabi-dopsis DGP2 (At4g10650) and DGP3 (At4g02790)proteins as well as proteins from rice, humans, Dro-sophila, Caenorhabditis, and Saccharomyces. Interest-ingly, some of the nonplant clade 1 proteins were shownto localize to mitochondria (Barrientos et al. 2003).SIN2 and DGP2, along with their respective riceorthologs Oryza SIN2 and Oryza DGP2, appeared tobe more closely related to each other than to any otherDAR proteins, suggesting that SIN2 and DGP2 resultedfrom a duplication postdating the divergence of plantsfrom other eukaryotes. Thus, it is possible that theseproteins share overlapping functions. DGP3, whichincludes a predicted chloroplast transit peptide (datanot shown), was placed with high bootstrap support intoa small subgroup within clade 1 containing the cyano-bacterial DAR proteins. Accordingly, DGP3 may play arole in the chloroplast that is analogous to that of SIN2.

DGP4 (At3g07050), DGP5 (At1g52980), and DGP6/7(At2g27200/At1g08410) were found in clades 2, 3,and 4, respectively, each of which also includes plant,animal, and fungal representatives, but no bacterialproteins. The clade 4 proteins DGP6 and DGP7 were72% identical and likely resulted from a relatively recent

duplication event. Only one clade 4 protein was foundfrom each of the other plant species represented in thesequence databases. Proteins within a particular cladeexhibited increased amino acid conservation amongresidues flanking each of the six described motifs. Clade2–4 proteins were longer than the clade 1 proteins atboth their N- and C-termini. The N-terminal extensionshad conserved features within each group. However, thesequences C-terminal to the LXG motif were highlyvariable both among and within groups.

Families of GTPases have been defined by similaritiesamong G1 and G2 sequence motifs (Caldon et al.2001). Our database searches and a previous study(Reynaud et al. 2005) indicated that the DAR subfamilyshares some sequence conservation with members ofthe Era family of GTPases, which include Era, EngA, andThdF/TrmE and which are found in all eubacteria(Caldon et al. 2001). A comparison of the SIN2 G1 andG2 motifs with the same regions from Escherichia coliEra family members showed that the G1 sequenceGXPNVGKS is conserved between SIN2 and Era familymembers (Figure 4B). The secondary structure pre-dicted from the DAR consensus sequence was comparedwith the E. coli EngA and Era crystal structures (Chen

et al. 1999; Robinson et al. 2002). All the G motifs of theDAR proteins were predicted to lie within similarstructural regions of EngA and Era (Figure 4D). Withinthis family, the DAR GTPases share the greatest se-quence similarity with EngA proteins. The G2 sequencePGXT was found in both SIN2 and EngA (Figure 4B;

Figure 3.—DAR protein alignment. An alignment of the seven Arabidopsis DAR proteins, SIN2 and DGP2–7, and the humanSIN2 ortholog, human DGP1, highlights the six conserved motifs of this family. Residues found in all of these proteins are shownin white on a black background. These include amino acids within the GTPase motifs G1–G4 and the DAR motif, labeled andunderscored below each sequence. There is also a region C-terminal to the recognizable GTPase motifs, which appears looselyconserved and includes a L(X)5G motif. Amino acids conserved between SIN2 and the closely related proteins DGP2 (38% iden-tity, 57% similarity) and human DGP1 (31% identity, 47% similarity) are shaded. Residues conserved between SIN2 and DGP2 orHsDGP1 are shown in black or white letters, respectively. The positions corresponding to the sin2-1 S150F transition and the sin2-2insertion are indicated above the sequence.


Caldon et al. 2001) and several EngA proteins werefound of have a D(X)6DAR motif 29 amino acids N-terminal to a G4-G1-G2-G3 segment (Figure 4C). Thisis similar to the 22-amino-acid distance between theSIN2 DAR and G4 motifs. In addition, the predictedDAR protein consensus structure had features similarto the region of EngA from its DAR-like motif to theC-terminal G3 (Figure 4D). These traits suggest that

SIN2 and other DAR GTPases may be derived from anEngA-like progenitor.

SIN2 localizes to mitochondria: The SaccharomycesDAR proteins have been found to localize to differentcellular compartments, including the nucleus, cytoplasm,and mitochondria (Bassler et al. 2001; Barrientos

et al. 2003). The subcellular localization of SIN2 wasevaluated in transgenic Arabidopsis plants expressing aSIN2:GFP fusion protein. The localization of the fusedGFP moiety was determined in the roots, hypocotyls,cotyledons, and trichomes of T2 seedlings. Most cellsshowed a punctate GFP fluorescence pattern thatcolocalized with fluorescent-stained mitochondria in-dicative of a mitochondrial localization of the SIN2protein in planta (Figure 5). This is consistent with thereported localization of the Saccharomyces and hu-man DAR proteins that group within the SIN2 clade 1(Barrientos et al. 2003).

DISCUSSION

SIN2 is a member of a bacterial/organellar subclassof the DAR GTPase family: Sequence similarity ana-lyses show that SIN2 includes the DAR motif and reveala noncanonical G-motif order in the DAR GTPases withthe G4 motif preceding G1–G3 (Figure 3; Fu et al. 1998).Structural similarities were found between the G domainsof DAR proteins and the E. coli Era GTPases, includingthe presence of conserved residues in the G1 and G2motifs (Figure 4B), suggesting that the DAR family isclosely related to the Era family (Caldon et al. 2001).

Figure 4.—Phylogenetic analysis of DAR GTPases. (A) Thesingle tree generated by parsimony analysis of a culled align-ment was modified to collapse branches that were not sup-ported in both culling and elision analyses by bootstrapvalues .50%. Four main clades are resolved, designatedclades 1–4. Eukaryotes are represented in each clade. Prokary-otic sequences are found in clade 1 and two orphan branches(including Methanococcus and Pyrobaculus sequences). Ara-bidopsis proteins are shaded and SIN2 is outlined in black.Numbers on the branches indicate bootstrap values basedon 500 bootstrap replicates. Branch lengths are relative to dis-tance as calculated using the transformed BLOSUM45 matrix.Clade designations are shown above the respective branches.Proteins without previous names were given ‘‘DGP’’ designa-tions. (B–D) DAR proteins share conserved sequences andsecondary structure with Era family members. (B) An align-ment of the G1 and G2 motifs of SIN2, E. coli Era, ThdF,EngA1, and EngA2 demonstrates that the SIN2 G1 motif issimilar to that of Era family members (Era, ThdF, and EngA).(C) A region of EngA similar to a DAR motif was foundbetween EngA1 G3 and EngA1 G4. (D) A comparison of theclade 1 consensus secondary structure predicted by the Jpredserver (Cuff et al. 1998) with that derived from the E. coliEngA and E. coli Era crystal structures (Chen et al. 1999;Robinson et al. 2002). EngA structure (arrows and cylindersindicate b-sheet and a-helix regions, respectively) appears toderive from a direct repeat of two Era-like units. The DARGTPases, represented by SIN2, resemble the central (G4–G3)region of the EngA proteins that include DAR motifs.


Interestingly, members of the EngA subclass of Eraproteins have two canonical G1–G4 GTPase domains intandem (Figure 4D; Mehr et al. 2000) and, therefore,contain the G4-G1-G2-G3 arrangement observed inDAR proteins. In addition, several EngA proteins in-clude a recognizable D(X)6DAR sequence motif at aposition relative to the first G4 motif that is similar tothat seen in DAR proteins (Figure 4C). Notably, there isadditional amino acid conservation between EngA andDAR proteins within the effector-binding G2 motif(Figure 4B). This level of conservation between EngAand DAR proteins suggests that the two classes derivefrom a common ancestor that included the duplicationof the canonical G1–G4 GTPase domains and a DARmotif and that the DAR proteins derived from this com-mon ancestor through truncation (Figure 4, B and D).

Eukaryotic DAR proteins resolve into four moderatelyto well-supported clades (1–4), suggesting an earlyexpansion of the gene family (Figure 4A; Reynaud

et al. 2005). In contrast, eubacterial DAR GTPases areconfined to clade 1, along with SIN2, DPG2, DGP3, andother eukaryotic representatives. This association issupported by another recent phylogenetic analysis(Reynaud et al. 2005). These results suggest that theeukaryotic proteins in this clade could have beenacquired in an early endosymbiotic event or otherhorizontal gene transfer. The first possibility is consis-tent with the mitochondrial localization of SIN2 (thiswork) and of its putative orthologs in yeast andmammals (Barrientos et al. 2003). The sequencesimilarity and close association among SIN2, DGP2,and plant orthologs in the phylogram indicates thatDGP2 is also likely a mitochondrial protein that derivedfrom a recent gene duplication event. In contrast, DGP3

and a rice ortholog associate with the DAR proteinsfrom known bacterial relatives of chloroplasts (Figure 4)and include putative chloroplast transit peptides (datanot shown). Thus, DGP3 likely represents the chloro-plast DAR ortholog in higher plants, as has beenpreviously hypothesized (Reynaud et al. 2005). Thestructure of clade 1 therefore suggests that each of thetwo endosymbiotic events related to plant evolution,acquisition of mitochondria and plastids, brought withthem specific DAR GTPase lineages.

DAR proteins are important for ribosomal assemblyin many subcellular compartments: Recent reportsshed light on the biological function of some of theclade 1 proteins that are putative orthologs of SIN2.Bacillus subtilis YlqF (Figure 4A) was shown to facilitatean essential step in the assembly of the large ribosomalsubunit (Matsuo et al. 2006; Uicker et al. 2006). Cellsdeficient in YlqF accumulate incomplete 50S ribosomalsubunits lacking the L16 and L25 protein components(Matsuo et al. 2006; Uicker et al. 2006). Evidenceof participation of the GTPase activity of YlqF in theassembly process is provided by the stimulation of YlqFGTPase activity in the presence of 50S subunits and byan association of YlqF with 50S subunits upon additionof a nonhydrolyzable GTP analog (Matsuo et al. 2006).Functional studies on humans and SaccharomycesMTG1 proteins indicate conservation of the ribosomeassembly function for these proteins in the eubacterial-derived mitochondria. Saccharomyces MTG1 was shownto be important for translation in mitochondria and arole in assembly of the mitochondrial large ribosomalsubunit was hypothesized (Barrientos et al. 2003). Itwas further shown that the human MTG1 protein couldpartially complement the Saccharomyces mtg1 mutant,indicating that the ribosome biogenesis function is likelyalso conserved in animals (Barrientos et al. 2003).

Other diverse DAR proteins have also been found toparticipate in ribosome biogenesis in other subcellularcompartments. The Saccharomyces clade 2 proteinNUG1 appears to be involved in ribosome assembly inthe nucleus and in transport through nuclear pores(Bassler et al. 2001; Nissan et al. 2002). The Saccharo-myces clade 3 protein NOG2 has functions similar toNUG1 and in addition may be involved in mRNAprocessing (Bassler et al. 2001; Saveanu et al. 2001;Bialkowska and Kurlandzka 2002). LSG1, the Sac-charomyces clade 4 protein, is located in the cytoplasmand is also important for ribosome transport throughnuclear pores (Nissan et al. 2002; Kallstrom et al.2003). This indicates conservation of a ribosome bio-genesis function among divergent DAR GTPase in thesubcellular compartments where they reside.

The reported functions of SIN2 orthologs as well asthe mitochondrial localization of SIN2 and of its closesteukaryotic orthologs suggests strongly that SIN2 is aDAR GTPase that functions in mitochondrial ribosomeassembly in Arabidopsis. SIN2 mRNA was present in all

Figure 5.—Localization of SIN2:GFP in Arabidopsis. Arabi-dopsis hypocotyl cells transformed with 35S:SIN2:GFP. Fluo-rescence appears white. (A) Green fluorescent protein islocalized to small particles within the cells that correspondto mitochondria stained with MitoFluor 589 dye, shown in B.


tested plant parts as would be expected for a mitochon-drial protein.

Effects of SIN2-reduced activity in Arabidopsis areconsistent with a role in supporting mitochondrialtranslation: At the molecular level, the mutation foundin the sin2-1 allele would generate a protein with a singleamino acid substitution in the GTP loop, the GTP-binding motif of the GTPase. Mutations in this motifwere identified in other GTPases and led to gain- or loss-of-function phenotypes (Bourne et al. 1991). However,the phenotypic overlap observed between the sin2-1 andthe insertional sin2-2 allele suggests that both allelescause loss of function or a decrease in SIN2 proteinactivity.

Mitochondria are involved in biochemical energyutilization and one would expect lower energy levelsto translate into reduced or retarded growth. Thereduced growth observed in the integuments of sin2ovules resulted from a decrease in the number of celldivisions in these structures (Broadhvest et al. 2000;this study). This reduced growth could result frominsufficient energy production due to compromisedmitochondrial activity. Indeed, we have previously char-acterized the phenotypic effects of mutations in HLL,which encodes a mitochondrial ribosomal protein ho-mologous to the L14 proteins of eubacteria (Skinner

et al. 2001). hll mutations result in a dramatic reductionin ovule growth, apparently due to compromised trans-lation in mitochondria (Schneitz 1999; Skinner et al.2001). The combination of sin2-1 and hll-1 mutationsis synergistic, leading to almost complete loss of ovuleprimordial growth (Broadhvest et al. 2000). This couldbe due to the combination of effects of the two muta-tions leading to an even more severe deficit in mito-chondrial function, at least during ovule development.Interestingly, the ant mutation was found to be epistaticto sin2-1 but synergistic with hll, leading to a similar butsomewhat less severe phenotype in comparison to thesin2-1 hll-1 double mutant (Broadhvest et al. 2000).This supports a hypothesis that ANT is needed to pro-mote growth and HLL is needed for growth to actuallyoccur. SIN2 would then facilitate the role of HLL inovule and integument growth. Thus, this suggests thatHLL is more important for mitochondrial activity thanSIN2, at least during ovule development, consistent withthe proposed structural and assembly roles for the twoproteins with respect to the large ribosomal subunit.

Many of the other phenotypic effects observed forsin2 Arabidopsis plants such as slower growth, reducedpollen formation, and reduced organ size are consistentwith a general decrease in energy metabolism thatwould be expected for plants with reduced mitochon-drial activity. However, the more complex phenotypesobserved also suggest a role for mitochondria in in-tegration of signaling pathways. For example, both main-tenance of axillary bud dormancy and continuity ofvascular tissue are under control of auxin-related path-

ways (Hardtke and Berleth 1998; Chatfield et al.2000). In addition, inhibition of auxin transport or mu-tation of the auxin response factor gene ETTIN causesdifferential effects along the length of the gynoeciumsimilar to the gradation of effects observed for sin2-2(Nemhauser et al. 2000). Thus, some aspects of SIN2action may reflect an interaction between phytohor-mone signaling and mitochondrial function. The broadrange of effects that sin2 mutations have on plantgrowth and architecture is consistent with the SIN2GTPase regulating mitochondrial activity in a variety ofdevelopmental pathways within the plant.

The observation that available sin2 mutants are notmore seriously growth deficient suggests that significantmitochondrial function likely persists in these mutantplants. This implies either that SIN2 activity is not alwaysrequired for assembly of the mitochondrial transla-tional apparatus or that a redundant activity exists. Sucha redundant activity could be supplied by DGP2, whichour sequence and phylogenic studies have identified asa late-branching paralog of SIN2. DGP2 could haveoverlapping function with SIN2 and could partiallycompensate for the reduction or loss of SIN2 activityin the sin2 mutants. Preliminary analyses of Arabidopsisdgp2 mutants show that this gene is functional and seemsto affect whole-plant architecture (T. Hill, unpublishedresults). This would parallel the observed redundancyfor HLL and the paralogous HULLENLOS PARALOG(HLP) gene (Skinner et al. 2001). More studies areneeded to evaluate the role of DGP2 in mitochondriaand putative paralogous interactions with SIN2.

Complexity in mitochondrial modulation of growthpathways: In plants, mitochondria have been shown tobe important for male fertility, appropriate expressionof floral homeotic genes, and programmed cell death(Christensen et al. 2002; Hoeberichts and Woltering

2003; Leino et al. 2003; Linke et al. 2003). Work in ourlaboratory has shown that at least two ribosomal pro-teins targeted to the mitochondria, HLL and SIN2, arenecessary for ovule development and that both alsoaffect the regulation of other developmental pathwaysin Arabidopsis. Interestingly, both of these genes havefunctional paralogs in Arabidopsis: DGP2 and HLP,respectively. Together, regulation of these genes couldallow for modulation of mitochondrial activity to facil-itate the differential growth necessary for morphogen-esis. The added complexity that cell mitochondrialnumber can vary in a tissue- and developmentally depen-dent manner provides additional plasticity for mito-chondria-integrated pathways. SIN2 and HLL, alongwith their paralogs, offer an entry point to understand-ing the regulation and impact of mitochondrial proteinsynthesis in plant development.

The authors thank Chris Roxas and Roderick Kumimoto fordedicated assistance in plant care and genotyping, Yuval Eshed forthe gift of pOCA28-15, the National Science Foundation ArabidopsisBiological Resource Center at Ohio State for Arabidopsis DNA clones,


and members of the Gasser and Bowman Labs for helpful discussions.This work was supported by the National Science Foundation (grantIBN-0079434) and by a U. S. Department of Agriculture NationalResearch Initiative Competitive Grants Program award (2001-35304-09989).

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