BREAKTHROUGH REPORT

OPENER Is a Nuclear Envelope and Mitochondria LocalizedProtein Required for Cell Cycle Progression in Arabidopsis[OPEN]

Wei Wang, Xueyang Zhang, and Totte Niittylä1

Umea Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183Umea, Sweden

ORCID IDs: 0000-0003-3400-4889 (W.W.); 0000-0002-3942-8333 (X.Z.); 0000-0001-8029-1503 (T.N.)

Currently one-third of the proteins encoded by the Arabidopsis (Arabidopsis thaliana) genome are of unknown function. Someof these unknown proteins are likely to be involved in uncharacterized vital biological processes. Evolutionarily conservedsingle copy genes in flowering plants have been shown to be enriched in essential housekeeping functions. This together withpublicly available gene expression data allows for a focused search for uncharacterized essential genes. Here we identify anessential single copy gene called OPENER (OPNR) in Arabidopsis. We show that OPNR is predominantly expressed in activelydividing cells and performs essential functions in seed development and root meristem maintenance. Cell cycle trackingusing 5-ethynyl-2’-deoxyuridine staining and fluorescent cell cycle markers together with the increased size of nucleolus andnucleus in opnr mutants indicate that OPNR is required for cell cycle progression through the S or G2 phases. Intriguingly,OPNR localizes to the nuclear envelope and mitochondria. Furthermore, the nuclear envelope localization of OPNR isdependent on its interaction with nuclear inner membrane Sad1/UNC-84 (SUN) domain proteins SUN1 and SUN2. Takentogether our results open a line of investigation into an evolutionarily conserved essential cellular process occurring in boththe nuclear envelopes and mitochondria of dividing cells.

INTRODUCTION

Essential genes are indispensable for the survival of an organismand constitute the minimum gene set required for cellular life.Many essential genes encode proteins of unknown function(PUFs). For instance, of the 473 essential genes in the synthesizedminimal bacterial genome, 149haveunknownbiological functions(Hutchison et al., 2016). In the model plant Arabidopsis (Arabi-dopsis thaliana), several independent large-scale genetic studieshave identified mutations in genes with an essential role in basiccellular processes. Thesemutations typically cause defects in theseed, zygote, or embryo phenotypes (http://www.seedgenes.org/; McElver et al., 2001; Meinke et al., 2008, 2009; Muralla et al.,2011; LloydandMeinke, 2012).Approximately15%(72outof481)of these essential Arabidopsis genes encode PUFs, and it is likelythat more essential genes await to be discovered among thecurrently 6838 Arabidopsis genes of unknownmolecular functioninvolved in an unknown biological process (www.arabidopsis.org). Investigation of PUFs is important because of their potentialto elucidate new biological processes and open new areas ofresearch. Reverse genetics investigations into genes andproteinsof completely unknown function are notoriously challenging.

However, it is possible to focus the search for the essential PUFsusing evolutionary clues and publicly available gene expressiondata combinedwith simple and fast assessment of seed-abortionphenotypes.Phenotype-guidedcharacterization of genes is often hampered

by the functional redundancy created by gene families. Genefamilies have their origins in gene and whole genome duplicationevents (Blanc and Wolfe, 2004; Van de Peer et al., 2009). Wholegenomeduplications and smaller scale gene duplications have animportant role in providing newmaterial for evolution (Ohno, 1970;Panchyetal., 2016). Interestingly, comparativegenomesequenceanalyses have also identified genes for which duplication appearstobedetrimental. Theseduplication resistant geneshave revertedto single copy status convergently in several plant species fol-lowing duplication events (Paterson et al., 2006; Duarte et al.,2010; De Smet et al., 2013). Such single copy genes encodeproteins for which two or more gene copies appear to be in someway disadvantageous (Paterson et al., 2006). Possible ex-planations include gene dosage linked gene transcript levelsensitivity, and cases where mutations in one copy are likely tocause dominant negative effects as is often the case for proteincomplexes (Veitia et al., 2008; De Smet et al., 2013). Analysis ofsingle-copy gene families in the genomes of 20 flowering plantsshowedenrichmentofessential housekeeping functions (DeSmetet al., 2013). Hence selection of evolutionary conserved singlecopy genes provides a way to narrow down the search for theessential genes encoding PUFs. Mutations in such single-copygenes can be expected to cause an obvious visible phenotypeearly in plant growth and development. In addition to new areas of

1 Address correspondence to totte.niittyla@slu.se.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Totte Niittylä(totte.niittyla@slu.se) and Wei Wang (wei.wang@slu.se).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00033

The Plant Cell, Vol. 31: 1446–1465, July 2019, www.plantcell.org ã 2019 ASPB.

research being opened, functional insights into such genes canalso shed light on the evolutionary forces behind the single-copygene selection observed in angiosperms (Paterson et al., 2006).

In this study, we identified an essential evolutionarily conservedsingle-copy gene OPENER (OPNR) that is required for cell pro-liferation in Arabidopsis. Mutation of OPNR results in zygote andendosperm arrest. The opnrmutants rescued by expression of thewild-type gene under a seed-specific promoter showed defects incell proliferation in the root tip meristem. In accordance with themutant phenotype OPNR is expressed in tissues with active celldivision. OPNR localizes to the nuclear envelope and physicallyassociates with the inner nuclear membrane Sad1/UNC-84 (SUN)domain proteins SUN1 and SUN2. Intriguingly, OPNR was alsolocalized to mitochondria. The dual localization of OPNR at thenuclear envelopeandmitochondria is unusual and theprotein lacksconserved functionaldomainssignifyingthatour resultsopenanewarea of research in cell cycle progression and plant growth.

RESULTS

Screening the Conserved Unknown Single Copy GeneSpace for Essential Genes

Even though Arabidopsis has been the subject of intensivefunctional genetics studies, ;30% of the proteins encoded bythe genome remain uncharacterized according to the latest ver-sion of the Arabidopsis Information Resource database (TAIR;Dhanyalakshmi et al., 2016). Some of these unknown genes canbe expected to be involved in processes which are essential forcellular life, and such essential genes can be postulated to beevolutionarily conserved in the green lineage.

To identify conserved, uncharacterized, and potentially es-sential genes in the Arabidopsis genome, we first listed the genesannotatedasbothunknownmolecular function (GO:0005554)andinvolved in unknown biological process (GO:0000004) according

to theTAIR10GeneOntologyAnnotations (www.arabidopsis.org;Berardini et al., 2004). The list contains 6838 genes, 5279 ofwhichare nuclear genes with an Arabidopsis Genome Identifyer locusnumber (Supplemental Data Set 1). To increase the likelihood ofobtaining amutant phenotype using reverse geneticswe selectedthe single copy genes from this list for further analysis. To identifygenes that are conserved in the green lineage, we used thecomparative genomics database PLAZA (https://bioinformatics.psb.ugent.be/plaza/; Van Bel et al., 2012) to find single genesconservedbetweenArabidopsis, themossPhyscomitrellapatens,and theunicellular greenalgaeChlamydomonas reinhardtii. Thesecriteria identified 194 unknown evolutionarily conserved singlecopygenes inArabidopsis (Supplemental DataSet 1). To focus ongeneswith a potential vital role in early plant development and cellproliferation, we analyzed gene expression data using Gene-vestigator (https://genevestigator.com/gv/; Zimmermann et al.,2004) and identified genes with predominant expression in theroot meristem (Supplemental Data Set 1).Mutations in essential genes required for fundamental cellular

processes often cause defects in the gametophyte or seed de-velopment; these defects typically manifest as reduced seed setand shorter siliques. Based on this concept we identified a T-DNAinsertion in one of the conserved single genes, which causeda very early seed abortion phenotype (Figure 1A). The T-DNA waslocated in the fifth exon of AT5G43822 (Figure 1A). The arrestedzygote in the mutant reminded us of a bottle opener (Figures 1Cand 1D), and therefore the gene was named OPENER (OPNR).Similarearlyseedabortionwasobservedforasecondallele,opnr-2,carrying a T-DNA insertion in the first intron of the same gene(Figure 1A; Supplemental Figure 1).

opnr Mutants Are Defective in Early Embryo andEndosperm Development

According to the TAIR10 database, the first intron of OPNR islocated in the 59-untranslated region (UTR), which also contains

Nuclear Envelope, Mitochondria, and Cell Cycle 1447

Figure 1. Characterization of the opnr T-DNA Insertion Lines.

(A) Schematic diagram of the OPNR gene structure and the T-DNA insertion sites. The gray and white boxes indicate translated and UTR of OPNR,respectively. The black box shows the location of AT5G43820 in the first intron of OPNR.(B) Developing seeds in the siliques from Col-0, opnr-1/1, opnr-1/1 3 Col-0, Col-0 3 opnr-1/1, and complemented opnr-1 plants 5 DAP. pO:Om,pOPNR:OPNR-myc transgene. White arrows indicate aborted seeds.(C)Ovule, zygote, and one-cell proembryo fromCol-0 and opnr-1plants. Black arrow indicates vacuole; ccn, central cell nucleolus; ecn, egg cell nucleolus;scn, synergid cell nucleolus.(D)Confocal laser scanningmicroscope images showautofluorescenceofCol-0 andopnr-1developing seeds. Elongated zygotes are indicated bydashedlines. Vacuoles and nuclei are indicated by yellow and blue arrows.(E) and (F) Areas of zygote nucleoli and stage III endosperm (4 endosperm nuclei per seed) nucleoli from Col-0 and opnr seeds, respectively. ***P < 0.001(unpaired t test, n 5 20).(G) Area of stage III endosperm nuclei from Col-0 and opnr seeds. ***P < 0.001 (unpaired t test, n 5 40).In (B), scale bars 5 0.5 mm; in (C) and (D), 10 mm.

1448 The Plant Cell

a coding sequence of another gene (AT5G43820). In earlierArabidopsis genome annotations, this locus encoded a singlegene (AT5G43820). To verify the current gene models, we ana-lyzed thecorresponding transcriptsbyamplifyingandsequencingdifferent sites from the two genes (Supplemental Figure 2A). Thefirst intron containing the AT5G43820 gene was spliced fromAT5G43822 59-UTR, and there was no detectable transcript in-cluding bothAT5G43820 andAT5G43822 (B). Two splice variantswere amplified for AT5G43822: the lower major band representsthe annotated coding sequence (AT5G43822.1), whereas the topweaker band contained the last intron of OPNR (AT5G43822.2;Supplemental Figure 2B).

To define the causal gene defect responsible for seed abortion,we performed complementation experiments using AT5G43820,AT5G43822.1, and AT5G43822.2 coding sequences under thecontrol of their native promoters. For the AT5G43822.1 andAT5G43822.2 complementation constructs, the start codon ofAT5G43820 located within the promoter sequence was mutatedfrom ATG to TTG. Only the AT5G43822.1 construct could rescuethe opnr-1/1 and opnr-2/1 seed abortion phenotype (Figure 1B;Supplemental Figure 1A; Supplemental Table 1), establishingAT5G43822 as the OPNR locus.

To characterize the seed abortion phenotype, we performedagenetic analysis of theopnrmutants (Table 1). Fromself-pollinatedheterozygous opnr-1/1 and opnr-2/1 plants, the ratios of progenywith opnrmutations to progenywithout opnrmutationswere 1.73:1and1.74:1 (opnr:wild type), respectively, deviating significantly fromthe expected Mendelian ratio of 3:1 (Table 1). Reciprocal crossesbetween the opnr mutants and wild-type plants showed that thetransmission efficiency of female opnr-1 and opnr-2 alleles wasreduced to 65%and 68% (Table 1), respectively, indicating that theopnrmutationsaffect femalegametophytedevelopment. Theopnr-1and opnr-2 male transmission efficiency was not affected (Ta-ble 1). Despite reasonable transmission of both male and femalemutant alleles, we could not obtain homozygous mutant plants inthe progenyof the heterozygous opnrmutants. This implies that inaddition to the femalegametophyte defect, theopnrmutantswerealso embryo lethal. Therefore, heterozygous mutant plants wereused for further phenotypic characterization.

To investigate the seed abortion phenotype in more detail,mature siliques from wild-type, self-crossed, and out-crossedopnr plants were dissected and observed under a stereomicro-scope. We counted aborted ovules and aborted seeds togetherbecause opnr mutant seeds aborted at a very early stage and it

was impossible to distinguish aborted seeds from aborted ovulesin mature siliques. The proportion of aborted ovules and seeds inwild-typesiliqueswas2.1%(n5704),whereas thecorrespondingvalues for heterozygous self-pollinated opnr-1/1 and opnr-2/1siliques were 31.5% (n5 620) and 28.7% (n5 745), respectively(Figure 1B; Supplemental Figure 1A; Supplemental Table 1).Moreover, when the opnr/1 pistils were pollinated with wild-typepollen, the proportion of aborted seeds in the resulting siliqueswas 16.0% (n5 650) to 16.5% (n5 675; Figure 1B; SupplementalFigure 1A; Supplemental Table 1). However, the abortion fre-quency was not significantly different from that for the wild type ifthe opnr/1 pollen was used to pollinate wild-type pistils(Figure1B;Supplemental Figure1A;Supplemental Table1).Theseresults were consistent with the genetic analysis indicating thatopnr mutations influence seed development.To further analyze the ovule and seed development defects,

unpollinated ovules and immature seeds from wild-type andmutant plants were excised, cleared, and examined with a dif-ferential interference contrast (DIC) microscope, which makesnucleoli more prominent than nuclei (Liu andMeinke, 1998). Mostof the nucleoli observed in the opnr ovules and young seeds wereclearly larger than those in the wild type (Figure 1C). At 2 d afteremasculation of stage 12 flowers, the mean areas of the centralcell nucleolus (ccn), egg cell nucleolus (ecn), and synergid cellnucleolus (scn) fromwild-type ovuleswere 39.064.8mm2, 10.061.3 mm2, and 8.36 0.9 mm2, respectively (n5 40). In opnr-1/1 andopnr-2/1 pistils, 49% (n 5 39) and 48% (n 5 40) of the ovulesexhibited comparable ccn areas (39.9 6 6.6 mm2 and 35.8 64.0 mm2, P > 0.05), but had clearly larger ecn (13.56 2.9 mm2 and12.4 6 1.7 mm2, P < 0.01) and scn (14.3 6 1.4 mm2 and 14.7 61.3 mm2, P < 0.01) areas. The remaining opnr-1/1 and opnr-2/1ovules had similar nucleolar areas to the wild type (for the ccn,36.56 5.8 mm2 and 39.66 4.9 mm2, P > 0.05; for the ecn, 10.060.9 mm2 and 10.6 6 1.4 mm2, P > 0.05; and for the scn, 8.4 61.2 mm2 and 8.76 1.8 mm2, P > 0.05). To determine whether theseovule defects affected pollination, seeds were cleared and ob-served at 2 d after pollination (DAP). The proportion of unfertilizedovules from opnr/1 self-pollinated siliques was 3.2% to 3.6%,whereas that from opnr/1 siliques pollinatedwithwild-type pollenwas 2.4% to 4.7% (Table 2). The ratio of unfertilized ovules fromopnr siliqueswasnot significantly higher than that for thewild type(1.7%unfertilizedovules, Table2). These results indicate thatopnrmutations led toenlargednucleoli in theeggcell andsynergidcellsin ovules, but did not affect pollination.

Table 1. Genetic Analysis of opnr Mutant Alleles

Crosses (Female 3 Male) With Insertion Without Insertion Ratio Expected Ratio TE, % x2

opnr-1/1 Self 71 41 1.73: 1 3: 1 NA 8.05a

opnr-2/1 Self 68 39 1.74: 1 3: 1 NA 7.48a

opnr-1/1 3 Col-0 117 180 0.65: 1 1: 1 65 13.36a

opnr-2/1 3 Col-0 44 65 0.68: 1 1: 1 68 4.05b

Col-0 3 opnr-1/1 83 76 1.09: 1 1: 1 100 0.31Col-0 3 opnr-2/1 72 70 1.02: 1 1: 1 100 0.03

Transmission efficiency (TE) 5 (progeny with insertion/progeny without insertion) 3 100; NA, not applicable; x2, Chi-square test statistic calculated forthe expected ratio.aResult differs significantly from the expected ratio (P < 0.01, one degree of freedom, x2c 5 6.63).bResult differs significantly from the expected ratio (P < 0.05, one degree of freedom, x2c 5 3.84).

Nuclear Envelope, Mitochondria, and Cell Cycle 1449

The most prominent phenotype of opnrmutants was observedduring early embryo and endosperm development. At 2 DAP,89.5% of seeds from wild-type plants were past the two-cellembryo stage and had a well-developed endosperm (Table 2).Conversely, only 54.8% to 55.7% of seeds from opnr/1 self-pollinated plants (hereafter referred to as opnr/1 seeds) and57.1% to 59.8% of seeds from opnr/1 siliques pollinated withwild-type pollen (hereafter referred to as opnr/1 3 Col-0[Columbia-0 wild-type] seeds) had reached a comparablestage (Table 2). The proportion of seeds lagging behind in theelongated zygote or one-cell embryo stages was 8.9% for wild-type seeds, 9.7% to 12.6% for opnr/1 seeds, and 21.4% to 24.5%for opnr/13 Col-0 seeds (Table 2). Moreover, 28.4% to 31.9% ofopnr/1 seeds and 13.7% to 16.4% of opnr/13Col-0 seeds werearrested at the zygote or one-cell embryo stageswith nomore thaneight endosperm nuclei, which was not observed in wild-typeseeds (Table 2; Supplemental Figures 2C and 2D). Similar de-fective embryo and endosperm development was also observed inopnr mutants at 3 DAP: most of the opnr-1 mutant seeds hadcollapsed by this point, but opnr-2 seed development was ar-rested slightly later (Supplemental Table 2).

When compared with wild-type seeds at similar stages of de-velopment, the opnr mutant seeds also exhibited enlarged nu-cleoli in both the arrested zygote and the endosperm (Figures 1Eand 1F; Supplemental Figure 1C). Using whole-mount DNAstaining, we found that also the endosperm nuclear size is in-creased in opnr seeds (Figures 1G). An elongated zygote witha large apical vacuole was observed in 7.20% to 7.60%of opnr/1seeds and 2.10% to 2.30%ofopnr/13Col-0 seeds, but not at allinwild-type zygotes (Table 2; Figure 1C;Supplemental Figure 1C).After fixation and clearing, the nucleoli, cytoplasm, and vacuolesof whole-mount developing ovules and seeds exhibit differentlevels of autofluorescence (Christensen et al., 1997). Basedon this, the apical vacuole phenotype was confirmed by confo-cal laser scanning microscope analysis of developing seeds(Figure 1D). Together these results indicate that the disruption ofOPNR caused nucleolus enlargement in egg cells, synergid cells,zygotes, early embryo cells, and the endosperm, and also causedzygote/embryo lethality and early endosperm arrest. In addition,

maternal OPNR expression is required for early embryo and en-dosperm development.

OPNR Is Predominantly Expressed in Tissues Active inCell Proliferation

Todetermine theexpressionpattern ofOPNR inArabidopsis, totalRNA was isolated from different tissues of wild-type Arabidopsisplants andOPNR transcript levels were determined by RT-qPCR.Aweakly expressed reference gene,UBIQUITIN-CONJUGATINGENZYME 21 (UBC21, AT5G25760; Czechowski et al., 2005), wasused for normalization. OPNR was expressed less strongly thanUBC, and its transcripts were present in every tested tissue(Figure 2A). To investigate the OPNR expression pattern in moredetail, a 4282-bp fragment upstreamof theOPNR start codonwasfused with the b-glucuronidase (GUS)-green fluorescent protein(GFP) reporter coding sequence and transformed into Col-0 plants. Three independent transgenic lines were analyzed, allofwhich showedGUSactivity in tissues active in cell division suchasshoot apexes, young leaves, root tips, and lateral root primordia(Figures 2B to 2I). These results indicate that OPNR is mainlyexpressed in proliferating tissues and also functions duringvegetative development.

OPNR Is Also Required During Vegetative Development

To investigate the function of OPNR in vegetative development,we performed an embryo rescue experiment using the FERTIL-IZATION INDEPENDENTSEED2 (FIS2) promoter (Luoet al., 2000)fused to the OPNR genomic sequence (pFIS2:OPNR). The pFI-S2:OPNR construct was introduced into opnr-1/1 plants andpartially rescued opnr-1 homozygous seedlings were obtained inthe progeny from 5 out of 43 independent transgenic lines. Theselines were named partially-rescued opnr (pr-opnr). The de-velopment of pr-opnr seedlings was abnormal and severelyretarded compared with the wild type (Figure 3A). RT-PCRexperiments showed that OPNR expression levels in pr-opnrseedlings were much lower than in the wild type (Figure 3B).SinceOPNR is predominantly expressed in proliferating cells, we

Table 2. Phenotypic Classes of opnr Alleles 2 DAP

Genotype 8c ;90en 4c ;48en 2c ;24en 1c$12en ez 12-16en 1c 1-8 en ez 6-8 en ez/z 1-4 en uf.o vc n

Col-0 self 5.0 46.0 38.5 7.5 1.4 0 0 0 1.7 0 3615 89.5 5 8.9 5 0 –

opnr-1/1 Self 1.1 24.9 28.8 8.6 1.1 1.7 5.0 25.2 3.6 7.2 3615 54.8 5 9.7 5 31.9 –

opnr-2/1 Self 5.0 29.6 21.1 12.0 0.6 2.6 5.3 20.5 3.2 7.6 3415 55.7 5 12.6 5 28.4 –

opnr-1/1 3 Col-0 1.1 21.2 37.5 19.3 2.1 1.9 5.1 9.4 2.4 2.1 3735 59.8 5 21.4 5 16.4 –

opnr-2/1 3 Col-0 1.0 25.1 31.0 21.4 3.1 2.6 3.9 7.2 4.7 2.3 3875 57.1 5 24.5 5 13.7 –

At 2 d after emasculation, stage 12 opnr/1 flowers were self-pollinated or pollinated with Col-0 pollen and the pistils were dissected 2 d later. Whole-mount ovules and seeds were cleared with Hoyer’s solution and observed under a DIC microscope. The percentage of each class is given underneaththe class. 8c, eight-cell embryo; 4c, four-cell embryo; 2c, two-cell embryo; 1c, one-cell embryo; en, endosperm nuclei; ez, elongated zygote; z, earlysymmetric zygote; uf.o, unfertilized ovules; vc, elongated zygote with vacuoles on the apical region; n, numbers of ovules and seeds from six siliques.Dashes indicate no data.

1450 The Plant Cell

focused our analysis of pr-opnr lines on the meristematic cells ofthe root tip. The size of the rootmeristem in iodine stained pr-opnrseedlings appeared smaller than the wild type at 5 d after ger-mination (DAG; Figure 3C). At 10 DAG, root hairs were formed atthe root tip of pr-opnr seedlings and the root cap starch granulesdisappeared (Figure 3C), which indicates that the root meristemcells had differentiated (Aida et al., 2004). These results showedthat OPNR is required for root meristem maintenance.

To investigate the cell cycle progression in root tips, wild-typeand pr-opnr seedlings were stained in situ with the thymidineanalog 5-ethynyl-2’-deoxyuridine (EdU) to analyze de novo syn-thesis of DNAduring theS-phase of the cell cycle (Kotogány et al.,2010). At 5 and 10 DAG, the nuclei of Col-0 root tips exhibitedtypical EdU staining (Figure 3D). In contrast, there were clearly

fewer stained nuclei in pr-opnr root tips at 5 DAG and no de-tectable stained nuclei at 10 DAG (Figure 3D). This indicated thatde novo DNA synthesis in pr-opnr root tips was impaired. After 10DAG the root tip cells of pr-opnr root tips were visibly swollen andhad lost root meristem activity (Figure 3D).To further analyze the cell cycle defect of the pr-opnr root tips,

we introduced anArabidopsis cell cyclemarker systemcalledCellCycle Tracking in Plant Cells (Cytrap; Yin et al., 2014) into the pr-opnr background. The Cytrap system expresses both the syn-thesis/Gap2 (S/G2) phase and endoreplicationmarkerpHISTONETHREE RELATED2 (HTR2):CDT1a (C3)–red fluorescent protein(RFP), and the late-G2/M phase marker pCyclin B1 (CYCB1):CYCB1-GFP (Yin et al., 2014). In the control Cytrap line, RFP-positive cells were detected throughout the root tip and were

Figure 2. OPNR Expression in Different Tissues and Organs.

(A)Expression levelsofOPNR indifferent tissuesanalyzedbyRT-qPCR.Youngsiliques,siliques1 to3DAP.Oldsiliques,siliques4 to8DAP.Threebiologicalreplicates are also shown.(B) to (H)Results of GUS staining showingOPNR promoter activity in a 5-d-old seedling (B), shoot apex (C), lateral root primordia at the hypocotyl and rootjunction (D), primary root tip (E), and different stages of lateral root formation [(F) to (I)].In (B), scale bars 5 2 mm; in (C) to (I), 50 mm.

Nuclear Envelope, Mitochondria, and Cell Cycle 1451

Figure 3. Partial Rescue of opnr Mutants Using the pFIS2:OPNR Construct.

(A) A Col-0 seedling 2 DAG and partially-rescued opnr (pr-opnr) seedlings 5 and 10 DAG, respectively.(B) RT-PCR showed that OPNR expression was drastically reduced in pr-opnr seedlings comparing with Col-0.(C) Lugol staining of Col-0 and pr-opnr roots 5 and 10 DAG, respectively.(D) Thymidine analog EdU staining of Col-0 and pr-opnr seedling root tips 5 and 10 DAG, respectively.(E)Wild-type andpr-opnr seedling root tips 5and10DAGexpressing the cell cyclemarker systemCytrap (Cell Cycle Tracking inPlantCells):pHTR2:CDT1a(C3)–RFP (RFP) and pCYCB1:CYCB1-GFP (GFP) that label cells in S/G2 and G2/M phase, respectively.In (A), scale bars 5 1 mm; in (C), 50 mm; in (D) and (E), 20 mm.

1452 The Plant Cell

often arranged into cell files. The GFP signals were observed inthe same region but in fewer cells (Figure 3E). These patterns arein line with published Cytrap results (Yin et al., 2014). In contrast,the root tips of pr-opnr seedlings contained a reduced number ofboth theRFP-positiveS/G2andGFP-positiveGap2/mitosis (G2/M) cells at 5 DAG (Figure 3E). There were no RFP or GFP positivecells in thepr-opnr seedlingsat 10DAG (Figure3E). These resultsare in line with the EdU staining and showed that cell cycleprogression is arrested in thepr-opnr lines. However, it is difficultto determine the exact stage of the cell cycle arrest in thepr-opnrroot tip cells, because pCYCB1:CYCB1-GFP marks cells fromlate G2 to metaphase (Yin et al., 2014). Nevertheless, theseresults suggested thatOPNR is required for cell proliferation andcell cycle progression.

OPNR Physically Associates with the Inner NuclearMembrane Proteins SUN1 and SUN2

OPNR is a 23-kD PUF with no obvious functional sequence do-mains. We hypothesized that identifying proteins associated withOPNR might clarify its function. To this end, a yellow fluorescentprotein (YFP) coding sequence was fused to the C terminus ofOPNR and expressed under the control of the native OPNRpromoter (pOPNR:OPNR-YFP). Thisconstruct complemented theopnr seed abortion phenotype (Supplemental Figure 1B), es-tablishing that the OPNR-YFP fusion protein was functional.OPNR-YFP coimmunoprecipitation (co-IP) experiments fromyoung seedlings did not precipitate sufficient amounts of proteinfor subsequent protein identification. Thiswas likely due to the lowlevel andmeristem-specificexpressionofpOPNR:OPNR-YFP. Toincrease the amount ofmeristematic cells and thereby the amountof extracted OPNR-YFP, we generated callus from the pOPNR:OPNR-YFP transgenic lines. The OPNR-YFP fusion protein andassociated proteinswere isolated frompOPNR:OPNR-YFP callususing GFP-Trap beads and analyzed by mass spectrometry.Free YFP isolated from p35S:YFP callus was used as a control.The mass spectrometry identified proteins are listed in theSupplemental Data Set 2.

Among the potential OPNR binding partners identified in thisway were the SUN domain proteins SUN1 and SUN2. SUN1 andSUN2 are nuclear envelope proteins involved in linking cyto-skeletal components with the nucleoskeleton, and sun1 sun2mutants exhibit meiotic defects (Varas et al., 2015). We thereforehypothesized that OPNR’s function may be linked to SUN-proteins, which would be consistent with the observation thatOPNR defects caused problems with cell cycle progression, andnuclear and nucleolar morphology in our phenotyping experi-ments. To confirm that OPNR physically associated with SUN1and SUN2 in vivo, we performed a co-IP assay using pOPNR:OPNR-YFP lines. p35S:YFP lines were used as negative con-trols. Total proteins were isolated and incubated with GFP–Trapbeads to immunoprecipitate OPNR-YFP and YFP, respectively,and anti-GFP and anti-SUN1-SUN2 antibodies were used toprobe the immunoprecipitated proteins. Anti-SUN1-SUN2 an-tibody specificitywasconfirmedby immunoblottingwith thewildtype and the SUN1 and SUN2 double knockout mutant, sun1-1;sun2-2 (Varas et al., 2015; Supplemental Figure 3A). As shown inFigure 4A, SUN1 and SUN2were detected in the immunoprecipitated

OPNR-YFP complex, but not in the YFP negative control, in-dicating that OPNR physically associates with SUN1-SUN2 inplanta.

OPNR-YFP Localizes to the Nuclear Envelope

The pOPNR:OPNR-YFP lines were also used to investigate thesubcellular localization of OPNR. In the root tip cells of thecomplemented opnr plants, the OPNR-YFP signal predominantlylocalized to the nuclear periphery and punctate structures in thecytosol, whereas a weak OPNR-YFP signal was also observedin the nucleoplasm and cytoplasm (Figure 4B). To increasethe resolution, we performed super-resolution imaging (ZeissLSM880 Airyscan) of OPNR-YFP signals in root cells. The imageshowed clear enrichment of OPNR-YFP signals on the nuclearperiphery and punctate cytosolic structures (Figure 4B). To de-termine whether the OPNR-YFP nuclear periphery signal colo-calized with SUN proteins, the inner nuclear membrane markerSUN2-cyan fluorescent protein (CFP;Oda andFukuda, 2011)wasintroduced intoOPNR-YFP lines. Imagingshowed that theOPNR-YFP signal on the nuclear periphery colocalized with SUN2-CFP(Figures 4C and 4D), confirming the nuclear envelope localizationofOPNRandsupporting theproposed interaction betweenOPNRand SUN1/2.To determine whether the nuclear envelope localization of

OPNR depends on SUN1 and SUN2, we introduced pOPNR:OPNR-YFP into the sun1-1; sun2-2 mutant background. OPNR-YFPdidnotconcentrateon thenuclear envelope in these lines,butdispersed in the cytosol and nucleoplasm (Figure 4E). To confirmthese results,we alsocrossedpOPNR:OPNR-YFP; sun1-1; sun2-2with Col-0 to generate pOPNR:OPNR-YFP; sun1-1/1; sun2-2/1plants. In these lines, OPNR-YFP was again observed on thenuclear envelope (Figure 4E). These results indicate that OPNRnuclear envelope localization depends on its association withSUN1 and SUN2.We also examined SUN1/2 localization in the pr-opnr lines by

introducing the SUN2-monomeric (m)RFP into wild-type and pr-opnr lines.TheSUN2-mRFPnuclearenvelope localizationwasnotaffected in the pr-opnr lines, but again we observed increasednuclear size in thepr-opnr root tips (Figures 4Fand4G). In additionwe also performed immunostainingwith an anti-SUN1/2 antibodyon wild-type and pr-opnr root tips. Although the antibody causedsomebackground immunostaining insun1-1; sun2-2 root tipcells,a typical SUN1-SUN2 nuclear envelop localization was observedin the Col-0 and pr-opnr lines (Supplemental Figure 3B). Theseresults showed that SUN1-SUN2 localization was not affected bythe opnrmutation. Notably, most of the sun1-1; sun2-2 seedlingsdid not show such obvious growth defects as the pr-opnr lines(Supplemental Figure 3C), which indicates that OPNR may haveother functions in addition to the SUN interaction-relatedprocesses.

OPNR Also Localizes to Mitochondria

OPNR-YFP signals also showed a punctate pattern in the cytosol(Figure4B). Todeterminewhether thesesignals are in the cytosol orsome cytosolic compartments, we transformed pOPNR:OPNR-YFP

Nuclear Envelope, Mitochondria, and Cell Cycle 1453

Figure 4. OPNR Is Associated with SUN1 and SUN2.

(A) Immunoprecipitation of OPNR-YFP and free YFP by GFP-trap beads from callus tissue. SUN1/2 were detected among the OPNR-YFP associatedproteins but not in experiments using the free YFP control. Two percent of the total protein extract used for IP was loaded as input.(B)OPNR-YFPsignalscapturedbynormalZeissLSM780confocal laser scanningmicroscope (top)orZeissLSM880Airyscansuper-resolutionmicroscope(bottom).(C) Confocal laser scanning microscope (CLSM) images of a nucleus of the root tip cell from a seedling stably expressing both pOPNR:OPNR-YFP andp35S:SUN2-CFP.(D) Fluorescence intensity profile along the line indicated by the arrow in (C).(E)ZeissLSM880Airyscansuper-resolutionmicroscope imagesshowingOPNR-YFP localization insun1-1; sun2-2andsun1-1/1;sun2-2/1backgrounds.(F) Expression of SUN2-mRFP in Col-0 and pr-opnr lines.(G) Nuclear area distribution of Col-0 and pr-opnr root tip cells. ***P < 0.001 (unpaired t test, n > 40).Scale bars 5 5 mm.

1454 The Plant Cell

lines with the p35S:pOPNR:mRFP construct. From the lines co-expressing OPNR-YFP and mRFP, it is clear that areas in thecytosol marked by OPNR-YFP were not overlapping with the freemRFP (Supplemental Figure 4A), indicating that OPNR-YFPsignals exist in a subcellular compartment that the free mRFPcannot enter. To investigate the identity of this compartment, wecrossed pOPNR:OPNR-YFP lines with marker lines expressingthe endoplasmic reticulum (ER) marker ER-CFP-HDEL (Pietraet al., 2013), Golgi marker SYNTAXIN OF PLANTS 32 (SYP32)-mCherry (Geldner et al., 2009), trans-Golgi network markerSYP61-CFP (Robert et al., 2008), and vacuolar marker RFP-C-terminal propeptide of phaseolin (CTPPpha; Hunter et al., 2007).However, none of these markers colocalized with OPNR-YFP(Figures 5A and 5B; Supplemental Figures 4B and 4C).

We also used organelle-specific dyes to study the cytosol lo-calization of OPNR-YFP. LysoTracker Red, which labels acidicvacuoles in plants (Merkulova et al., 2014), did not colocalize withOPNR-YFP signals (Supplemental Figure 4D). The amphiphilicstyryl dye FM4-64, which first stains the plasma membrane (PM)and then labels the vesicle system from PM to the vacuole (Rigalet al., 2015), did not show obvious colocalization with the OPNR-YFP in the cytosol after 1 to 4 h of labeling (Supplemental Figures4E and 4F). A Brefeldin A (BFA) treatment blocks trafficking ofendosomes to the PM and generates BFA bodies in the cell (Rigalet al., 2015). pOPNR:OPNR-YFP lines treated with BFA and FM4-64 showed BFA bodies stained with FM4-64, but OPNR-YFPsignals did not colocalize with BFA bodies (SupplementalFigure 4G). Taken together, these colocalization experimentsshowed that OPNR-YFP cytosol signals are not localized in theendomembrane system including the ER, Golgi, trans-Golginetwork, and vacuoles.

To further investigate the OPNR-YFP localization, we per-formed MitoTracker Deep Red staining and observed clear co-localization of the punctate cytosolic OPNR-YFP signals withMitoTracker staining (Figure 5C). Control experiments withpOPNR:OPNR-YFP lines without staining and wild-type linesstainedwithMitoTracker ruled out thepossibility of bleed-throughof fluorescence emission (Supplemental Figures 4H and 4I). Tofurther validate theMitoTracker stainingspecificity,westained theroot tipsofamitochondriamarker linemit-GFP (LoganandLeaver,2000) with MitoTracker (Thermo Fisher Scientific), and the resultsshowed thatmit-GFPsignalsmatchwellwithMitoTracker staining(Supplemental Figure 4J). Interestingly, in the sun1-1; sun2-2background OPNR-YFP did not concentrate on the nuclear en-velopebut still colocalizedwithMitoTracker (Figure5D), indicatingthat the mitochondria localization of OPNR is independent of theSUN1/2 interaction. In linewith theMitoTracker colocalization, theOPNR-YFP coimmunoprecipitated proteins included two mito-chondria localized proteins, PROHIBITIN3 (PHB3) and PHB4(Supplemental Data Set 2; Van Aken et al., 2007). To test whetherthe OPNR and PHB3 colocalize to the same compartment andcould therefore interact, we introduced a native promoter drivenPHB3 fused with mCherry (pPHB3:PHB3-mCherry) to pOPNR:OPNR-YFP lines. OPNR-YFP and PHB3-mCherry showed clearoverlap on mitochondria (Figure 5E).

To assess the subcellular dynamics of OPNR-YFP during thecell cycle, the OPNR-YFP/SUN2-CFP line was crossed witha mRFP-TUB6 tubulin marker line (Ambrose et al., 2011).

Individualcell cyclephaseswereassociatedwithspecificchangesin the localization of SUN2 (Oda and Fukuda, 2011) and micro-tubules (Chabouté and Schmit, 2001). The OPNR-YFP signalswere present both in the nuclear envelope andmitochondria frominterphase to early mitotic prophase (Supplemental Figure 5), butfromprometaphase to anaphase, it was primarily found only in themitochondria (Supplemental Figure 5). In early telophase, OPNR-YFP labeled mitochondria were detected around the nuclearenvelope,and in late-telophaseOPNR-YFPcolocalizedagainwithSUN2-CFP to the nuclear envelope of the daughter cells(Supplemental Figure 5). These results established that OPNR-YFP is found in themitochondria duringmetaphase andanaphaseafter the breakdown of the nuclear envelope and nucleolus, andassociates with the nuclear envelope in late telophase as thenuclear envelope and nucleolus are reformed.

Mitochondria Size Is Reduced in the pr-opnr Root Tip Cells

To investigate the effect of lack of OPNR on the nuclear envelopeand mitochondria, the morphology of nuclear envelope and mi-tochondria in the pr-opnr root tip cells was examined by trans-mission electron microscopy (TEM). The structure of nuclearenvelope showed no obvious difference between pr-opnr cellsand the wild-type control (Figure 5G). There was also no cleardefect in the structure ofmitochondria in the pr-opnr root tip cells.However, the average size and overall size distribution of mito-chondria was reduced in the pr-opnr root tip meristematic cells atboth 5 and 10 DAG (Figures 5G and 5F). Notably the proportion oflarger mitochondria (area > 0.3 mm2) in the pr-opnr is significantlylower than in thewild type.Themitochondriasizedistribution in theroot tip cells of pr-opnr is closer to the one observed in the dif-ferentiated lateral root cap cells of wild type with big vacuoles(Figure 5F). In addition, we observed large vacuoles in the pr-opnrrootmeristematic cells at 10DAGbut not at 5DAG (Figure 5G).Wefurther measured and calculated the percentage of mitochondriaarea of the total cytosol area excluding nucleus. The relativemitochondria area in pr-opnr root tip meristematic cells at 5 DAG(10.2%61.9%, n5 10 cells) is comparable with wild-type root tipmeristematic cells at both 5DAG (12.3%63.6%, n5 10 cells) and10DAG (11.5%62.6%,n510cells). In contrast, themitochondriaarea ratio in the wild-type lateral root cap cells is clearly lower(5.0%61.1%, n5 10 cells). The mitochondria ratio in the pr-opnrroot tipmeristematic cells at 10DAG (7.2%62.2%, n5 10 cells) isbetween that of wild-type root tip meristematic cells and lateralroot cap cells. Dumbbell-shape mitochondria are typically in-terpreted as dividing mitochondria (Jiang et al., 2006; Kuroiwaet al., 2006). The ratio of dumbbell-shapemitochondria of pr-opnrroot tipmeristematic cells at 5DAG (14.5%,n5503mitochondria)is slightly higher than that ofwild-type root tipmeristematic cells at5DAG (11.0%n5109mitochondria) and10DAG (10.6%,n5199mitochondria). However, the ratio of dumbbell-shape mitochon-dria in pr-opnr cells at 10 DAG (5.2%, n 5 251 mitochondria) issimilar to that of wild-type lateral root cap cells (5.0%, n 5 120mitochondria). These results indicate that thepr-opnr root tip cellshad differentiated at 10 DAG but not yet at 5 DAG. The smallermitochondria in the pr-opnr root tip meristematic cells at 5 DAG,therefore, supports a direct function of OPNR in mitochondria.

Nuclear Envelope, Mitochondria, and Cell Cycle 1455

Figure 5. Subcellular Localization of OPNR-YFP in Mitochondria.

(A) and (B) CLSM images of root tip cells from seedlings stably expressing pOPNR:OPNR-YFP and SYP32-mCherry (A) and SYP61-CFP (B).(C) and (D) MitoTracker Deep Red staining of root tip cells from seedlings stably expressing pOPNR:OPNR-YFP in opnr-1 (C) and sun1-1; sun2-2 (D)background, respectively.(E) CLSM images of root tip cells from seedlings stably expressing pOPNR:OPNR-YFP and pPHB3:PHB3-mCherry.

1456 The Plant Cell

DISCUSSION

We identified an evolutionarily conserved essential Arabidopsisprotein of unknown function, named OPNR here, which localizesto nuclear envelope and mitochondria. opnr mutants show earlyseed abortion due to arrested embryo and endosperm de-velopment (Figure 1C; Table 2). Partial rescue of opnr in the seedsby the FIS2 promoter allowed us to investigate the function ofOPNR in young seedlings. The TEM images of pr-opnr rootmeristem cells at 10 DAG contained cells with large vacuoles(Figure 5G), reminiscent of the large apical vacuoleobserved in theopnr zygote (Figure 1C). The opnr zygote and pr-opnr meriste-matic cells stopped dividing and possibly underwent cell differ-entiationasevidentby theextensionof roothairs into the root tipofpr-opnr seedlings (Figure 3B), and the large vacuoles and reducedmitochondria size in the root meristem (Figures 5F and 5G).Combined with the EdU staining and Cytrap results, whichshowed fewer stained nuclei and reduced number of mitotic cells(Figures 3D and 3E), we propose that OPNR is required for cellcycle progression. To resolve the exact stage of the cell cyclearrest in the opnrmutants requires further experiments. However,the enlarged nucleoli in the opnr zygote and endosperm (Figures1C to 1E) and the larger nuclei in the opnr endosperm and pr-opnrroot tips (Figures 1G, 4F, and 4G) indicate an arrest at the S or G2stageof thecell cycle. This conclusion is basedon theobservationthatnuclear size increasesduring theSphaseprovidingaproxy forcell cycle stages (Jovtchev et al., 2006; Chen et al., 2017).

TheOPNR-YFP signal is concentrated on the nuclear envelope,and immunoprecipitation experiments established physical in-teraction of OPNR with the integral inner nuclear membraneproteins SUN1 and SUN2 during interphase and prophase,suggesting a function in nuclear envelope-related processes(Figure 4; Supplemental Figure 5). The nuclear envelope consistsof the inner nuclear membrane, outer nuclear membrane, andnuclear pore complexes. In addition, a meshwork of nuclearlamins is present beneath the inner nuclear membrane, providingmechanical strength to thenucleus inmetazoans (GruenbaumandFoisner, 2015;Meier et al., 2017).Plant genomes lackhomologsoftheanimal lamins, but lamin-likefilamentousmeshworkstructuresand lamin-like proteins have been found in plants (Masuda et al.,1997; Dittmer et al., 2007; Fiserova et al., 2009). SUN1 and SUN2are type II transmembrane proteins resident in the inner nuclearmembrane. Their N termini are located in the nucleoplasm andassociate with lamin-like proteins, whereas their C termini containthe SUN domain and associate with outer nuclear membraneKlarsicht/ANC-1/Syne-1 Homology (KASH)-domain proteins thatinteract with the cytoskeleton. SUN and KASH domain proteinsform theLINCcomplex,which connects the nucleoskeleton to thecytoskeleton (Meier et al., 2017; Gumber et al., 2019). Previousstudies have suggested that LINC complexes are involved in

regulating nuclear movement, nuclear positioning, nuclear mor-phology, and chromatin–nuclear envelope interactions (Starr andFridolfsson, 2010; Tapley and Starr, 2013). Animal SUN proteinsare known to be involved in various processes including main-taining nuclear and nucleolar morphology, meiosis, mitosis, DNAdamage responses, and mRNA export (Lei et al., 2012; Turgayet al., 2014; Li and Noegel, 2015; Lawrence et al., 2016;Matsumoto et al., 2016).Plant SUN proteins have been studied less extensively than

their animal counterparts. The Arabidopsis genome encodes twoclassical c-terminal SUN domain proteins (SUN1 and SUN2) andthree atypical mid-SUNs (SUN3, SUN4, and SUN5) with a SUNdomain in the center of the protein (Graumann et al., 2014).Mutations in SUN-encoding genes lead to several developmentaldefects. When compared with wild-type plants, the sun1-1knockout mutant and the sun1-KO; sun2-KD double mutant(Oda and Fukuda, 2011; Zhou et al., 2015a, 2015b, 2015c) pro-ducedmore spherical root hair nuclei.Moreover, theSUN1-SUN2double knockout mutant sun1-1; sun2-2 showed severe meioticdefects (Varas et al., 2015).Whencomparedwithwild-typeplants,the sun3 mutant produced more spherical root epidermal cellnuclei and sun4 sun5 double mutant plants produced smallernuclei (Graumann et al., 2014). Deletion of all three mid-SUNproteins caused early seed abortion (Graumann et al., 2014).These results suggest that like animal SUNs, plant SUNs haveimportant functions on nuclear morphology control and plantdevelopment.OPNRcontainsnopredicted transmembranedomains (datanot

shown), suggesting that it is recruited to the nuclear envelope byits interactionwithSUN1-SUN2.Thiswas further supportedby theobservation that OPNR-YFP signals do not concentrate on thenuclear envelope in sun1-1; sun2-2 mutants (Figure 4E). HenceOPNR associates with SUN1-SUN2, but whether they directlyinteract with each other remains an open question. OPNR-YFPcolocalized with SUN2-CFP during interphase and prophase, butthe fluorescence signals showed no obvious overlap after nuclearenvelope breakdown (Supplemental Figure 5). It is thus possiblethat the interaction between OPNR and SUN1-SUN2 is mediatedby other proteins or requires modifications that occur during thecell cycle. On the other hand, SUN1-SUN2 localization is notaffectedby theopnrmutation (Figure4F;Supplemental Figure4B),showing that OPNR is not involved in SUN1-SUN2 localization tothe inner nuclear membrane.The meiotic defects of SUN1/2 double knockout mutants (Oda

andFukuda,2011;Zhouetal., 2015a,2015b, 2015c) arenot seen inopnr mutants. This may be because residual OPNR transcripts orthe corresponding proteins from parental heterozygous diploidsporocytes enable mutant gametophytes to survive before fertil-ization (Muralla et al., 2011). On the other hand, embryo de-velopment and vegetative growth inSUN1-SUN2 double knockout

Figure 5. (continued).

(F) Size distribution of mitochondria in root tip cells from pr-opnr and Col-0 at different stages. The areas of at least 200mitochondria from at least 10 cellswere analyzed for each case. ***P < 0.001 (unpaired t test, n > 200).(G) TEM images of Col-0 and pr-opnr root tip meristematic cells 5 DAG and 10 DAG. Bottom, images are close up view of mitochondria from top images.(H) TEM images of a Col-0 lateral root cap cell. m, Mitochondria.In (A) to (E), scale bars 5 5 mm; in (G) and (H), 500 nm.

Nuclear Envelope, Mitochondria, and Cell Cycle 1457

mutants is less severely impaired than in opnr mutants(Supplemental Figure 3C; Oda and Fukuda, 2011; Zhou et al.,2015a, 2015b, 2015c). It may be that other mid-SUN proteins(SUN3, SUN4, and SUN5) can compensate for the function ofSUN1/2 in plants. Mid-SUNs are also localized to the ER andnuclear envelope, and interact with KASH domain proteins(Graumann et al., 2014). In keeping with this hypothesized func-tional redundancy among SUN proteins, sun1-1; sun4-1; sun5-1mutants showedamore severe phenotype than sun1-1; sun4-1orsun4-1; sun5-1 doublemutants (Poulet et al., 2017), and knockingoutall threemid-SUNs inArabidopsis results inearlyseedabortion(Graumann et al., 2014).

Further clues on the function of OPNR SUN1/2 interactioncomes from the OPNR-YFP co-IP experiments. The potentialOPNR interactors included a Cell Division Cycle 48 (CDC48)homolog protein CDC48D and another CDC48-like protein CAMINTERACTING PROTEIN 111 (CIP111) identified with the highsequence coverage (Supplemental Data Set 2). These two pro-teins belong to the ATPase associated with a variety of cellularactivities (AAA) superfamily. In conjunction with cofactors/adaptors, CDC48 proteins couple ATP hydrolysis and so calledsegregase activity, which removes ubiquitinated target proteinsfrom protein complexes, organelle membranes, or chromatin fordegradation or recycling (Bègue et al., 2017; Bodnar and Rapo-port, 2017; van den Boom and Meyer, 2018). Hence, a hypo-thetical OPNR function is as a cofactor/adaptor of the CDC48proteins for substrate recognition and recruitment. SUN1-SUN2could potentially be substrates for such CDC48-OPNR-mediatedprotein turnover or segregation. In support of this idea SUN2proteins in mammalian cells were identified as the substrates ofthe Skp Cullin F-box (SCF)bTrCP1/2 E3 ubiquitin ligase (Coyaudet al., 2015; Kim et al., 2015). SUN2 was stabilized by b-TrCPknockdown and accumulation of SUN2 proteins affected nuclearmembrane integrity (Coyaud et al., 2015). SUN proteins form verystable complexes with KASH domain proteins but how the in-teraction is modified is not known (Sosa et al., 2013). SUN-KASHinteractions are disrupted when nuclear envelope breaks downduring cell cycle. OPNR could potentially be involved in segre-gating SUN1-SUN2 from SUN-KASH complexes and inner nu-clear membrane for either degradation or recycling. The absenceof OPNR may affect SUN1-SUN2 protein interactions and/orturnover and result in nuclear envelope and cell cycle defects.However, there are several phenotypic differences between opnrand sun mutants, which leads us to hypothesize that OPNRperformsadditional vital functionsashintedby itsdual localizationat the nuclear envelope and mitochondria (Figures 4 and 5).

Mitochondria are highly dynamic semi-autonomous organellesthat undergo constant fission and fusion, which is thought tocontribute to sharing of mitochondrial DNA, recombination repairand replication of mitochondrial DNA, distribution of mitochon-drial components, removal of damaged components and selec-tive mitochondria autophagy. The balance between fission andfusion ofmitochondria can change the number, size, and shape ofmitochondria and affect plant development and stress responses(Logan, 2010; Liberatore et al., 2016;Møller, 2016; Arimura, 2018).In general, mitochondria play an important role in cell prolifera-tion in plants, and disruption of mitochondria-related genesoften shows meristem defects. One example is the Arabidopsis

ATP-dependent zinc metalloprotease FtsH4, which is a mito-chondrial inner membrane-integrated protease with proteolyticdomains facing the intermembrane space. The ftsh4mutant losesstem cell identity at elevated temperature (30°C), presumably dueto accumulation of oxidatively damaged proteins (Dolzblasz et al.,2016, 2018). Another example is Arabidopsis SLOW GROWTH3,which encodes a pentatricopeptide repeat protein for splicing ofintron 2 of mitochondrial NADH dehydrogenase subunit 7 (NAD7)gene. The slo3 mutants show severe growth retardation and re-duced root apical meristem size (Hsieh et al., 2015). Anotherpentatricopeptide repeat protein ABA OVERLY SENSITIVE 8(ABO8) that is responsible for the splicing of intron 3 of mito-chondria NAD4 gene also has a role in controlling root meristemactivity (Yang et al., 2014). The nuclear envelope localization ofOPNR-YFP was lost in the sun1-1; sun2-2 (Figure 5D), which hasa less severe phenotype compared with pr-opnr (SupplementalFigure 3C). This could indicate that the critical function of OPNR isassociated with mitochondria.There isnopredictedmitochondrial signal peptide, nopredicted

transmembranedomain,or tail-anchoredmotif in theOPNRaminoacid sequence (data not shown). Hence, similar to the SUN1-SUN2-mediated nuclear envelope localization, OPNR maylocalize to mitochondria through protein-protein interaction. In-terestingly, the co-IP experiment identified two mitochondrialprohibitin proteins PHB3 and PHB4 among the possible OPNRinteractors (Supplemental Data Set 2). Prohibitins are integralmitochondrial inner membrane proteins that form 1–2-MDa ring-like protein complexes, which are proposed to act as scaffold/chaperones to stabilize other protein complexes such as matrix-ATPases associatedwith a variety of activities (m-AAA) proteasesand respiration chain complexes I andV.Previous studies showedthat PHB3 and PHB4 are prominently expressed in meristemtissues and phb3 null mutants exhibit meristematic cell pro-liferation defects (Van Aken et al., 2007, 2010; Piechota et al.,2010; Kong et al., 2018). We showed that PHB3-mCherry co-localizes with OPNR-YFP on mitochondria (Figure 5E), whichtogether with the co-IP, similar meristem expression, and rootmeristem defects, support a functional relationship betweenOPNR and PHB3 and PHB4 in mitochondria.Mitochondrial function of OPNR was also supported by the

reduction in the mitochondria size distribution in pr-opnr root tipsboth at 5 and10DAG (Figures 5F and5G).Mitochondria are highlydynamic organelles that undergo constant fission and fusion andthe balance between fission and fusion of mitochondria canchange thenumber, shapeandsizeofmitochondria (Logan, 2010;Møller, 2016; Arimura, 2018). Abnormal mitochondria size isusually associated with mitochondria fission or fusion defects.Mutations in genes involved inmitochondrial fission usually resultin elongated or enlargedmitochondria. For example, mutations inplant dynamin-related protein 3A (DRP3A) and DRP3B causeelongated, network-shaped mitochondria (Fujimoto et al., 2009).Defects in mitochondrial outer membrane anchored proteinsArabidopsis thaliana Fisson 1a (AtFis1a) and AtFis1b (BIGYIN1and BIGYIN2) also cause larger mitochondria (Scott et al., 2006;Zhang and Hu, 2009). Disruption of the plant specific proteinELONGATED MITOCHONDRIA 1, which is required for the lo-calization of DRP3A also cause elongated mitochondria (Arimuraet al., 2008). In contrast, defects in genes for mitochondrial fusion

1458 The Plant Cell

alwaysresult inmitochondriaaggregationor fragmentation. Inyeast(Saccharomyces cerevisiae) and animals, mitochondrial fusion iscontrolled by two kinds of dynamin related GTPases, FuZzy On-ions1 (Fzo1)/mitofusin (Mfn) and mitochondrial genome mainte-nance1 (Mgm1)/Optic atrophy1 (Opa1), which are outer and innermembrane-integrated proteins, respectively (Arimura, 2018). Mu-tants lackingFzo1orMgm1showfragmentedmitochondria (Sesakiand Jensen, 1999; Sesaki et al., 2003). Proteins involved in mito-chondria fusion in plants are largely unknown (Arimura, 2018).FRIENDLY, a protein from the CLUSTERED MITOCHONDRIAsuperfamily, was proposed to play a role in intermitochondrialassociation before fusion andmutation of it results inmitochondriaaggregation (Logan et al., 2003; El Zawily et al., 2014).

In plants, mitochondria change their shape and size accordingto tissue type, development stage, cell cycle, programmed celldeath, and external stimuli. Tubular and reticularmitochondria arefound in germinating seeds, shoot apical meristem, and regen-erating protoplasts (Sheahan et al., 2005; Seguí-Simarro et al.,2008; Seguí-Simarro and Staehelin, 2009; Paszkiewicz et al.,2017). Indifferentiated andsenescencecellsmitochondria usuallyshow round, oval, or sausage-like morphology and smaller size(Zottini et al., 2006; Welchen et al., 2014; Liberatore et al., 2016).We found that in wild-type Arabidopsis there are relatively largermitochondria in the root tip meristematic cells compared with thedifferentiated lateral root capcells. The reducedmitochondria sizein the pr-opnr lines may be a direct effect of OPNR disruption ora consequence of the cell cycle arrest and subsequent cell dif-ferentiation. The latter idea is supported by the similar mito-chondria size distribution observed between the pr-opnr root tipsanddifferentiated lateral root cap cells ofwild type (Figures 5Fand5H). However, it is likely that OPNR plays a direct role in mito-chondria as discussed above.

Dissecting the temporal dynamics of cell cycle arrest and mi-tochondrial defects in pr-opnr root tips will require further work.However, the TEM micrographs taken at 5 and 10 DAG togetherwithpublishedstudiesofmitochondrial phenotypesprovidesomehints. First, at 10DAG, therearenodividingcells in thepr-opnr roottip cells (Figure 3) and TEM observation of these root tip cellsshowed features of differentiated cells including large vacuolesand reduced size and number of mitochondria (Figure 5H). At 5DAG there are still dividing cells in the pr-opnr root tips (Figure 3),and the TEMobservation of these cells did not show big vacuolesas observed for the differentiated lateral root cap cells (Figure 5H).Furthermore, in pr-opnr root tips at 5 DAG the mitochondria sizewas reduced, but thepercentageofmitochondria areaper cell andthe percentage of dumbbell-shape mitochondria are similar towild-type meristematic cells. This was in contrast to the differ-entiated lateral root cap cells where both the mitochondria areaand the dumbbell-shape mitochondria percentages are reduced.These observations suggest that at 5 DAG the mitochondria sizereduction is likely a direct effect ofopnrdisruption, and not a resultof cell differentiation. Second, the phb3mutants, which also havecell proliferation defects in the root tip, have mitochondria abouttwo times larger than wild type (Van Aken et al., 2007). This in-dicates that cell cycle arrest does not necessarily lead to mito-chondria size reduction.

An additional clue for OPNR function in mitochondria comesfromtheOPNR-YFPco-IPexperiments that identifiedmitochondria

proteins PHB3/4 and two AAA type ATPasesCDC48D andCIP111(Supplemental Data Set 2). Animal PHBs were found to be ubiq-uitinated during spermiogenesis for elimination of paternal mito-chondria (Thompsonetal., 2003;Houetal.,2017).Recentstudies inyeast showed that CDC48 proteins play a role in mitochondriafusionby regulatingFzo1ubiquitylationanddynamics.Mutations inCDC48 cause mitochondria aggregation and fragmentation (Esakiand Ogura, 2012; Chowdhury et al., 2018; Simões et al., 2018). Inaddition, CDC48 is also involved in degradation of other mito-chondrial outermembraneproteinssuchasMdm34,Msp1,Tom70,andoxidationdamagedmitochondrial proteins in yeast andMcl1 inhuman (Xuetal.,2011;Hemionetal., 2014;Wuetal., 2016).Hence itis possible that OPNR acts as a cofactor of CDC48 proteins inmitochondria protein dynamics and quality control to regulatemitochondria size and function.The dual localization of OPNR at both nuclear envelope and

mitochondria is unusual. Only a fewproteins so farwere implied tohave similar dual localization. Examples include the human sar-colemmalmembrane-associated protein (SLMAP), which is a tail-anchored protein conserved from yeast to human. SLMAP isa subunit of the striatin-interacting phosphatase and kinasecomplex and plays a role in a variety of cellular processes such ascell cycle progression and chromosomal inheritance (Nordziekeet al., 2015). PRO45 is the striatin-interacting phosphatase andkinase homolog in filamentous fungi Sordaria macrospora andstudies indicate that enhanced green fluorescent protein (EGFP)fused PRO45 is found on the nuclear envelope, endoplasmicreticulum, and mitochondria (Nordzieke et al., 2015). It was pro-posed that PRO45 might bridge the nuclear envelope and themitochondria and act as a membrane organizer to mediate sig-naling (Nordzieke et al., 2015). Another example is the Caeno-rhabditis elegans cell death protein 4 (CED-4), which is normallylocated tomitochondria by bindingwith the anti-apoptotic proteinCED-9. Upon cell death induction, CED-4 is disassociated fromCED-9 and binds to the caspase CED-3 for its activation. Duringthis process, CED-4 is released from mitochondria and trans-located to the nuclear envelope in a CED-3–independent manner(Chen et al., 2000). Interestingly a follow-up study found that theinner nuclear membrane protein SUN1 is the nuclear envelopereceptor for CED-4 (Tzur et al., 2006). It was suggested that thenuclear envelope localization of CED-4 targets the apoptoticmachinery to nuclei destined for degradation (Tzur et al., 2006).SLMAP/PRO45 or CED-4 are examples of nucleus and mito-chondria localized proteins, but they lack homologs in plants anddo not provide clues to OPNR function.It seems plausible that OPNR functions in a protein complex on

the membranes of nucleus and mitochondria, possibly as a co-factor/adaptor of the CDC48 proteins for recognition and bindingof substrate proteins on nuclear envelope and mitochondria. Aprotein complex function is also supported by the fact thatOPNRhas been selected as a single copy gene in the green lineage(Supplemental Data Set 1). As mentioned in the introductionprotein complexes are prone to dominant negative mutationevents, the likelihoodofwhich is lower for singlecopygenes (Veitiaet al., 2008; De Smet et al., 2013). Further work on OPNR maycontribute to the understanding of the mechanisms driving geneloss during evolution. At this point OPNRopens a completely newline of investigation into a previously uncharacterized essential

Nuclear Envelope, Mitochondria, and Cell Cycle 1459

process occurring in both nucleus and mitochondria individing cells.

METHODS

Plant Material and Growth Conditions

TheArabidopsis (Arabidopsis thaliana) ecotypeColumbia (Col-0)was usedas a wild-type control. Seeds of Arabidopsis T-DNA insertion lines (Col-0)were obtained from the Nottingham Arabidopsis Stock Centre: opnr-1(SALK_148287) and opnr-2 (GK-556C04). The T-DNA insertion site wasdetermined by sequencing PCR products with gene-specific primers andT-DNA border primers. Mutant and wild-type plants were grown in soil at22°C with a photoperiod of 16-h light and 8-h dark and 65% relative hu-midity.ValoyaNS12 lightemittingdiode (LED) tubeswereusedand the lightintensity is150mmolm22 s21.pOPNR:OPNR-YFPandp35S:YFPcalliweregeneratedasdescribedbyCheetal., 2006.All primersused in thisstudyarelisted in Supplemental Table 3.

Phenotypic Analysis

Siliques fromwild-type andmutant plantswere dissectedwith fineneedlesandovulesorseedswereexcised intact andmountedwithHoyer’ssolution(Liu and Meinke, 1998). After 1 to 6 h of clearing, samples were examinedunder a Axioplan 2 microscope (Zeiss) equipped with DIC optics anda AxioCam HRc camera (Zeiss). Images were processed and analyzedusing ZEN2011 (Zeiss) and ImageJ (http://www.imagej.nih.gov/ij/). Con-focal laser scanning microscope analysis of developing seeds was per-formed as described previously (Christensen et al., 1997). Siliques werecollected and fixed in 4% (w/w) glutaraldehyde and 12.5 mM cocadylate(pH 6.9) under vacuum for 2 h. Then the siliques were dehydrated ina graded ethanol series (20% steps for 1 h each), and cleared in a 2:1mixture of benzyl benzoate:benzyl alcohol. The siliquesweremounted andobserved under a LSM780 confocal laser scanning system (Zeiss)mountedonanAxioobserverZ1 invertedmicroscope (Zeiss)with theeGFPsetting. Open siliques and seedlings were observed and photographedunder a MZ 9.5 stereomicroscope (Leica).

DAPI (4’,6-diamidino-2-phenylindole) staining was performed as de-scribed previously (Street et al., 2015), with some modifications. Ara-bidopsis wild-type and mutant seedlings were fixed with 4% (w/v)formaldehyde in PEMT buffer (50 mM PIPES, 2 mM EGTA, 2 mM MgSO4,and 0.05% [v/v] Triton X-100, pH 7.2) for 40 min. Fixed seedlings wererinsed with PEMT buffer three times and then washed with phosphate-buffered saline (PBS) buffer three times (5 min for each wash). Seedlingswere then stainedwith 20 ng/mLDAPI in PBSbuffer for 10min, followedbythree washes in PBS buffer. Seedlings were mounted in PBS buffer, andDAPI signals were visualized under a LSM780 confocal laser scanningmicroscope (Zeiss). The nuclear circularity index was taken to equal 4pA/P2

(A, cross-sectional area of the nucleus; P, perimeter of the nucleus).Toexamine endospermnuclear size in developingseeds fromwild-type

and opnr plants, we performed whole-mount seed DNA staining as pre-viously described by Baroux et al., 2007.

EdU in situ staining was performed using the EdU Alexa Fluor� 488Imaging Kit (Thermo Fisher Scientific) as described previously (Kotogányet al., 2010). Briefly, seedlingswere treatedwith 20mMEdU for 1 h and thenfixed in fixation solution (4% [w/v] formaldehyde, 0.1% [v/v] Triton X-100 inPBS buffer, pH 7.5) for 30 min. The fixation solution was removed, and theseedlingswerewashed3 timeswithPBSbuffer (10minperwash). Then theseedlings were incubated with EdU detection cocktail in darkness for30 min. After 3 washes with PBS buffer (10 min each), root tips weremounted and observed under a LSM780 confocal laser scanning micro-scope (Zeiss).

Lugol staining of starch deposition in the root tips was performed asdescribed inHongetal., 2015. The rootsofCol-0andpr-opnrseedlingsat5DAG and 10 DAG were submerged in Lugol solution (Sigma-Aldrich) for1min. The roots were then rinsed twice with distilled water andmounted inclearing solution (80 g chloralhydrate, 30mL glycerol, and 10mLwater) for2 h. Then the roots were visualized with a Axioplan 2 microscope (Zeiss)equipped with DIC optics.

RNA Expression Analysis

Total RNAwas extracted fromArabidopsis tissues using the RNeasy PlantMini Kit (Qiagen) according to the manufacturer’s instructions. Contami-nating DNA in RNA was removed with the Ambion DNA-free Kit (ThermoFisher Scientific) and then cDNA was synthesized using the iScript cDNASythesisKit (Bio-Rad).RT-qPCRwasperformedonaBio-RadCFX96Real-Time systemwith iQSYBRGreenSupermix (Bio-Rad). Aweakly expressedreference gene, UBC21 (AT5G25760; Czechowski et al., 2005), was usedfor normalization. There are 2 biological replicates and 3 technical repli-cates for eachbiological replicate. RT-PCRwasperformedwithDreamTaqDNA Polymerase (Thermo Fisher Scientific) on a Bio-Rad S1000 Touchthermal cycler. Thirty cycles were performed for OPNR coding sequenceamplification, and 26 cycles were performed for ACT2 amplification.

Complementation and Partial Rescue of the opnr Mutants

For complementation experiments with OPNR, the 4281 bp promotersequence upstream of the OPNR start codon was amplified and the startcodon ofAT5G43820 in this fragment wasmutated fromATG to TTGusingPCR-based site-directed mutagenesis (Heckman and Pease, 2007). TheOPNR promoter and OPNR coding sequence (AT5G43822.1 andAT5G43822.2) were fused in the pDONR207 vector (Thermo Fisher Sci-entific) by Gibson cloning (New England Biolabs) to create the pOPNR:OPNR.1 and pOPNR:OPNR.2 entry vectors. The pOPNR:OPNR.1 andpOPNR:OPNR.2 sequences in the entry vector were transferred to thedestination binary vector pEarleyGate303 (Earley et al., 2006) by LR re-action (ThermoFisher Scientific) to generate thepOPNR:OPNR.1-myc andpOPNR:OPNR.2-myc constructs. For complementation withAT5G43820,the 4281-bp sequence upstream of the OPNR start codon was amplifiedand fused in the pDONR207 vector by Gibson cloning, and transferred tothe destination binary vector pKGWFS7 by the LR reaction. These con-structs were introduced into opnr-1/1 and opnr-2/1 mutant plants usingAgrobacterium tumefaciens-mediated floral dip transformation (Cloughand Bent, 1998). Plants homozygous for both T-DNA insertions andtransgenic genes (opnr-1/opnr-1;OPNR.1-myc/OPNR.1-myc and opnr-2/opnr-2; OPNR.1-myc/OPNR.1-myc) were obtained in the T3 generation.The seeds from the transgenic opnr homozygous plants were observedunder a MZ 9.5 stereomicroscope (Leica).

For partial rescue experiments, the OPNR genomic sequence wascloned into a pENTRY/D-TOPO vector (Thermo Fisher Scientific) and thentransferred to pFIS2 destination vector (Roszak and Köhler, 2011) togenerate the pFIS2:OPNR binary vector by the LR reaction (Thermo FisherScientific). The pFIS2:OPNR construct was introduced into opnr-1/1mutant plants by Agrobacterium-mediated transformation (Clough andBent, 1998). Plants homozygous for both T-DNA insertions and transgenicgenes (opnr-1/opnr-1; pFIS2:OPNR/pFIS2:OPNR) were obtained in the T3generation. The wild-type and partially-rescued opnr (pr-opnr) seedlingswere observed under a MZ 9.5 stereomicroscope (Leica) and a Axioplan 2microscope (Zeiss).

Protein Localization

The pOPNR:OPNR entry vector and the pHGY (RIKEN Plant ScienceCenter, Japan) destination vector were used to create the pOPNR:OPNR-

1460 The Plant Cell

YFP construct by the LR reaction (Thermo Fisher Scientific). Genomicfragments corresponding to SUN2 were amplified and cloned into thepDONR207 vector (Thermo Fisher Scientific) by Gibson cloning (NewEngland Biolabs) to create the SUN2 entry vector. Then the SUN2 entryvector and the pEarleyGate102 and pK7RWG2 destination vectors wereused to create the p35s:SUN2-CFP and p35S:SUN2-mRFP construct bytheLRreaction (ThermoFisherScientific). Formaking thep35S:pOPNR:mRFPconstruct, the 4281-bp sequence upstream of the OPNR start codon wasamplified and fused in the pDONR207 vector by Gibson cloning, andtransferred to the destination binary vector pK7RWG2 by the LR reaction.

For making the pPHB3:PHB3-mCherry construct, the promoter andcoding sequence of PHB3 (AT5G40770) gene without stop codon, themCherry coding sequence, the rbcS terminator (Outchkourov et al., 2003),and the backbone sequence of pGreen 0029 vector (Hellens et al.,2000) were amplified and assembled by Gibson cloning (New EnglandBiolabs). ThepOPNR:OPNR-YFP,p35S:pOPNR:mRFP,p35s:SUN2-CFP,p35S:SUN2-mRFP, and pPHB3:PHB3-mCherry constructs were in-troduced into Arabidopsis plants by Agrobacterium-mediated trans-formation (CloughandBent, 1998). The linescoexpressingpOPNR:OPNR-YFP and marker lines ER-CFP-HDEL (Pietra et al., 2013), SYP32-mCherry(Geldner et al., 2009), SYP61-CFP (Robert et al., 2008), RFP-CTPPpha

(Hunter et al., 2007), ormRFP-TUB6 (Ambrose et al., 2011) were generatedby crossing. For the observation of these lines, seedlings were grownvertically on plates containing half Murashige and Skoog (MS) media with0.8% (w/v) plant agar for 5 d and then observed under a Zeiss LSM780 orLSM880 confocal laser scanning microscope. For Airycan imaging ofOPNR-YFP signals, the Zeiss LSM880 confocal laser scanning micro-scope with Airyscan and a LD LCI Plan-Apochromat 403/1.2 Imm Auto-Corr DIC M27 water immersion objective was used. The super resolutionmode of theAiryscan detectorwas used, and scaling per pixel is 0.05 mm3

0.05 mm. The excitation wavelength is 514 nm, and emission was detectedby filterBP495-550nmandLP570nm.The raw imageswereprocessedbyusing the standard 2D Airyscan filtering in the ZEN black software.

Immunostaining of SUN1/2 proteins was performed as described pre-viously (Boutté andGrebe, 2014). Briefly, seedlingswere grown vertically onhalf MS agar plates for 5 d and then fixed in freshly prepared 4% (w/v)paraformaldehyde in microtubule-stabilizing buffer (MTSB, 50 mM PIPES,5 mM EGTA, 5 mM MgSO4, pH 7) for 1 h at room temperature. The fixedseedlings were washed twice with MTSB (10 min each) and 3 times withdouble distilled water (10 min each). Then root tips were cut, placed onsuperfrost slides (Thermo Fisher Scientific), and left to dry at room tem-perature for ;10 min. The dried slides were sealed with double-sided tapeand cover slips, and then the samples on the slides were rehydratedwithMTSB for 15min and incubatedwith 2% (w/v) Driselase (Sigma-Aldrich)inMTSB for 30minat 37°C.Root tipsonslideswerewashed three timeswithMTSBandthenpermeabilizedwithpermeabilizationbuffer (10%[v/v]DMSO,1%[v/v] IGEPALCA-630 inMTSB[Rhodia]) for1hat roomtemperature.Roottipswerewashed3 timeswithMTSBand incubatedwithblockingbuffer (2%[w/v] bovine serum albumin [BSA]inMTSB) for 1 h at room temperature. Theprimary antibody (rabbit anti-SUN1/2, AS15 2856, Agrisera AB; 1:200 inblocking buffer) was then added and the samples were incubated at 4°Covernight and foranextra2hat37°C.The root tipswere thenwashed4 timeswith MTSB, incubated with the secondary antibody (DyLight594 Donkeyanti-rabbit, ab96921, Abcam; 1:300 in blocking buffer) for 2.5 h at 37°C,washed 3 times with MTSB, and then incubated with DAPI staining buffer(20ng/mLDAPI inMTSB) for10minat roomtemperature.Afterbeingwashed3 times with MTSB, the cover slips were removed and the root tips weremounted in a drop of Citifluor AF1 (Electron Microscopy Sciences) andobserved under a 780 confocal microscope (Zeiss).

LysoTraker, MitoTracker, and FM4-64 Staining

pOPNR:OPNR-YFP seedlings were incubated with 5 mMLysoTracker RedDND-99 (Thermo Fisher Scientific) or 200 nM MitoTracker Deep Red FM

(Thermo Fisher Scientific) in half MS liquid media for 30 min at roomtemperature. After staining, the seedlings were rinsed twice with half MSand observed under a LSM780 confocal laser scanning microscope(Zeiss). For FM4-64 staining, pOPNR:OPNR-YFP seedlings were im-mersed in 10 mMFM4-64 (Thermo Fisher Scientific) in liquid half MSmediafor 1 to 4 h. For BFA (Thermo Fisher Scientific) treatment, 50 mMBFA wasapplied together with FM4-64 dye.

GFP-Trap Immunoprecipitation Followed by Immunoblot or TrypsinOn-Bead Digestion

Total proteins from p35S:YFP and pOPNR:OPNR-YFP calli were ex-tracted with extraction buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mMEDTA; 0.5% [v/v] Triton X-100; 0.5% [v/v] IGEPAL CA-630; 10% [v/v]glycerol; 1 3 Complete protease inhibitor cocktail [Roche]) and in-cubatedwith GFP-Trap-MA (Chromotek) beads for 1 h at 4°C. The beadswere then washed three times with wash buffer (50 mM Tris-HCl, pH 7.5;150 mM NaCl; 10% [v/v] glycerol; 0.3% [v/v] Nonidet P-40, and 1 3

Complete protease inhibitor cocktail). For immunoblotting, proteins onbeads were eluted by boiling in 1 3 laemmli sample buffer (Bio-Rad) at95°C for 5 min. The eluted proteins were resolved by SDS-PAGE,transferred to a PVDF membrane (Immobilon-P, Millipore), probed withmouse anti-GFP (JL-8, Clontech, 1:2000) or rabbit anti-SUN1-SUN2antibodies (AS15 2856, Agrisera AB; 1:1000), and detected with theSuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo FisherScientific). For on-bead trypsin digestion, the beads were washed anadditional three timeswith50mMNH4HCO3 (pH7.4), treatedwithDTT (finalconcentration 5 mM, in 50 mM NH4HCO3) for 15 min at 90°C, and thenalkylated (iodoacetamide, final concentration 10mM) at room temperaturefor 30 min in darkness. Then trypsin was added and the sample was di-gested for 30 min at 27°C. After the initial digestion, the magnetic beadswere removed with a magnet rack and the supernatant was collected intoa fresh tube, in which it was left to stand overnight at 37°C for furtherdigestion. The reaction was then quenched with 1% (v/v) trifluoroaceticacid. The protein digest was desalted and concentrated on Oasis HLBcartridges (Waters, 96-well mElution Plate, 5 mg sorbent), after which theeluate was evaporated to dryness in a Mi-Vac Centrifugal Vacuum Con-centrator (SP Scientific). Formic acid (0.1% [v/v]) was used to reconstitutesamples before mass spectrometry analysis.

Mass Spectrometry and Data Analysis

The sample was injected onto a nano-flow ultra performance liquidchromatography-tandem mass spectrometer (UPLC-MS/MS) systemconsisting of a nanoACQUITYTM ultra-performance liquid chromatog-raphy system (Waters) connected to a Synapt G2-Si Quadrupole-Ionmobility-TOF high definition mass spectrometer (Waters). The samplewas first loaded onto a trap column (ACQUITYUPLCM-Class Trap, C18,5 mm, 180 mm i.d. 3 20 mm; Waters) at 15 mL/min and then eluted andseparated on an HSS T3 C18 column (75 mm i.d. 3 150 mm, 1.8 mmparticles; Waters) using a linear 145min gradient of 0.5%-40% solvent B(75% [v/v] Acetonitrile, 25% [v/v] Isopropanol). The column was thenbalancedwith99%solventA (0.1%[v/v] formicacid inwater) at aflowrateof 300 nL/min. The separated sample was introduced into the Synapt G2mass spectrometer via a nanoflowelectrospray ionization (ESI) interfaceoperating in positivemode. All data were collected in continuumHDMSEmode with a drift time-specific collision energy (Distler et al., 2014) andmass-corrected using Glu-fibrinopeptide B and Leu Enkephalin as ref-erence peptides.

The rawdatawere used for database searchingwithProteinLynxGlobalServer (PLGS, Waters). For Arabidopsis, the TAIR-10 database was used.Peptide and fragment mass tolerances were set at 10 ppm and 25 ppm,respectively, and the number of allowed mis-cleavages was set to 1.

Nuclear Envelope, Mitochondria, and Cell Cycle 1461

Carbamidomethylation at Cwas set as a fixmodification, whereas variablemodifications were N-terminal acetylation, deamidation at N or Q, oxi-dation at M, and phosphorylation at S, T or Y. FDR was set at 1%.

TEM Sample Preparation and Observation

Col-0 and pr-opnr root tips were cut and fixed by high pressure freezing in0.15MSucusingaLeicaHPM100.Frozensampleswere freezesubstitutedusing the LeicaAFS2 system for 33 h in acetone, 0.5% (w/v) uranyl acetate,and 3% (v/v) water and thenwarmed up to245°C and during a linear warmup to225°C embedded inHM20. The 70-nm sections were postcontrastedwith 5% (w/w) uranyl acetate and Reynolds lead citrate and examined witha Talos 120C (FEI) operating at 120kV. Micrographs were acquired witha Ceta 16M charge-coupled device camera (FEI) using TEM Image andAnalysis software ver. 4.17 (FEI).

Accession Numbers

The genes andmutants used in this study are: OPNR (AT5G43822), SUN1(AT5G04990), SUN2 (AT3G10730), PHB3 (AT5G40770), PHB4(AT3G27280), CDC48D (AT2G03670), CIP111 (AT3G56690), UBC21(AT5G25760), ACT2 (AT3G18780), opnr-1 (SALK_148287), opnr-2 (GK-556C04), sun1-1 (SAIL_84_G10), and sun2-2 (FLAG_026E12).

Supplemental Data

Supplemental Figure 1. Seed development phenotypes of opnrmutants and complementation.

Supplemental Figure 2. Confirmation of OPNR gene model inArabidopsis.

Supplemental Figure 3. SUN1/2 antibody verification, immunostain-ing of SUN1/2 proteins and sun1-1; sun2-2 seedlings.

Supplemental Figure 4. Subcellular localization of OPNR-YFP andcell compartment markers.

Supplemental Figure 5. Subcellular localization of OPNR-YFPthroughout the cell cycle.

Supplemental Table 1. Seed abortion ratio of opnr mutant andcomplementation lines.

Supplemental Table 2. Phenotypic classes of opnr alleles 3 days afterpollination.

Supplemental Table 3. List of primers used in this study.

Supplemental Data Set 1. Identification of arabidopsis evolutionarilyconserved single copy genes with enriched expression in the rootmeristem.

Supplemental Data Set 2. OPNR-YFP and free YFP co-IP and massspectrometry data.

ACKNOWLEDGMENTS

WethankSusanArmstrong forproviding thesun1-1; sun2-2doublemutantseeds andMasaaki Umeda for sharing the Cytrap seeds. For the help withTEM experiments, we thank Sara Henriksson from the Umeå Core Facilityfor Electron Microscopy at Umeå University and the National MicroscopyInfrastructure (VR-RFI 2016-00968) for providing assistance in micros-copy. This work was supported by grants from the Swedish ResearchCouncil Formas (Svenska Forskningsrådet Formas), Bio4Energy (SwedishProgramme for Renewable Energy), and the Umea Plant Science Centre,

Berzelii Centre for Forest Biotechnology funded by VINNOVA. X.Z. wassupported by a postdoc fellowship from the Kempe Foundation.

AUTHOR CONTRIBUTIONS

W.W. conducted the experiments; X.Z. conducted themass spectrometryanalysis; W.W. and T.N. designed experiments, analyzed data, and wrotethe article.

Received January 17, 2019; revised April 1, 2019; accepted April 23, 2019;published April 25, 2019.

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DOI 10.1105/tpc.19.00033; originally published online April 25, 2019; 2019;31;1446-1465Plant Cell

Wei Wang, Xueyang Zhang and Totte NiittyläProgression in Arabidopsis

OPENER Is a Nuclear Envelope and Mitochondria Localized Protein Required for Cell Cycle

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