BREAKTHROUGH REPORT

Leaf Variegation and Impaired Chloroplast DevelopmentCaused by a Truncated CCT Domain Gene inalbostrians Barley[OPEN]

Mingjiu Li,a,b Goetz Hensel,c Martin Mascher,d Michael Melzer,e Nagaveni Budhagatapalli,c Twan Rutten,e

Axel Himmelbach,a Sebastian Beier,f Viktor Korzun,g Jochen Kumlehn,c Thomas Börner,b and Nils Steina,1

aGenomics of Genetic Resources Group, Department of Genebank, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK),06466 Seeland, GermanybMolecular Genetics Group, Institute of Biology, Humboldt University, 10115 Berlin, Germanyc Plant Reproductive Biology Group, Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop PlantResearch (IPK), 06466 Seeland, GermanydDomestication Genomics Group, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), 06466 Seeland, Germanye Structural Cell Biology Group, Department of Physiology and Cell Biology, Leibniz Institute of Plant Genetics and Crop PlantResearch (IPK), 06466 Seeland, Germanyf Bioinformatics and Information Technology Group, Department of Breeding Research, Leibniz Institute of Plant Genetics and CropPlant Research (IPK), 06466 Seeland, GermanygKWS LOCHOW, 29303 Bergen, Germany

ORCID IDs: 0000-0003-3299-5287 (M.L.); 0000-0002-5539-3097 (G.H.); 0000-0001-6373-6013 (M.Ma.); 0000-0002-5213-4030(M.Me.); 0000-0002-5537-8276 (N.B.); 0000-0001-5891-6503 (T.R.); 0000-0001-7338-0946 (A.H.); 0000-0002-2177-8781 (S.B.);0000-0003-0760-8249 (V.K.); 0000-0001-7080-7983 (J.K.); 0000-0001-9548-3348 (T.B.); 0000-0003-3011-8731 (N.S.)

Chloroplasts fuel plant development and growth by converting solar energy into chemical energy. They mature fromproplastids through the concerted action of genes in both the organellar and the nuclear genome. Defects in such genesimpair chloroplast development and may lead to pigment-deficient seedlings or seedlings with variegated leaves. Suchmutants are instrumental as tools for dissecting genetic factors underlying the mechanisms involved in chloroplastbiogenesis. Characterization of the green-white variegated albostrians mutant of barley (Hordeum vulgare) has greatlybroadened the field of chloroplast biology, including the discovery of retrograde signaling. Here, we report identification ofthe ALBOSTRIANS gene HvAST (also known as Hordeum vulgare CCT Motif Family gene 7, HvCMF7) by positional cloning aswell as its functional validation based on independently induced mutants by Targeting Induced Local Lesions in Genomes(TILLING) and RNA-guided clustered regularly interspaced short palindromic repeats-associated protein 9 endonuclease-mediated gene editing. The phenotypes of the independent HvAST mutants imply residual activity of HvCMF7 in the originalalbostrians allele conferring an imperfect penetrance of the variegated phenotype even at homozygous state of the mutation.HvCMF7 is a homolog of the Arabidopsis (Arabidopsis thaliana) CONSTANS, CO-like, and TOC1 (CCT) Motif transcriptionfactor gene CHLOROPLAST IMPORT APPARATUS2, which was reported to be involved in the expression of nuclear genesessential for chloroplast biogenesis. Notably, in barley we localized HvCMF7 to the chloroplast, without any clear evidence fornuclear localization.

INTRODUCTION

Chloroplasts are the site of photosynthesis. In vascular plants,functional chloroplasts differentiate from their progenitor organ-elles called proplastids. This process, chloroplast biogenesis,depends on a network of environmental, temporal, and cellular

factors (Pogson and Albrecht, 2011), with the latter including theexpressionofbothplastidandnucleargenes.Thenucleargenomecodes for the vast majority of proteins required for chloroplastbiogenesis. In barley (Hordeum vulgare), for instance, only 78 of;3000 chloroplast proteins are encoded in the plastid genome(plastome; Saski et al., 2007; Petersen et al., 2013). Mutations inthesenucleargenesmay result indefectiveplastidsas reflectedbyfrequently occurring leaf coloration aberrations in different plantspecies. Chlorophyll-deficient mutants provide valuable genetictools for the identification of nuclear genes involved in chloroplastbiogenesis and regulation (Taylor et al., 1987; Barkan, 1998; Leonet al., 1998).Specifically, mutations leading to variegation, due to their

unique feature of exhibiting both normal and defective plastids in

1 Address correspondence to: thomas.boerner@rz.hu-berlin.de or stein@ipk-gatersleben.de.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Nils Stein (stein@ipk-gatersleben.de).[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00132

The Plant Cell, Vol. 31: 1430–1445, July 2019, www.plantcell.org ã 2019 ASPB.

different sectors of the same tissue, are of great interest toresearch toward understanding (1) chloroplast biogenesis, (2)cross-communication between the nucleus and the other DNA-containing compartments such asplastids andmitochondria, and(3) themolecular mechanism of variegation. Genes involved in thevariegation pattern formation have been cloned in monocot anddicot species (Wu et al., 1999; Chen et al., 2000; Takechi et al.,2000; Prikryl et al., 2008; Hayashi-Tsugane et al., 2014; Wanget al., 2016; Zheng et al., 2016; Guan et al., 2017; Zagari et al.,2017). Despite the advances of identifying the underlying genes,insights into the molecular mechanisms controlling the phe-nomenon are rare (Sakamoto, 2003; Yu et al., 2007). Leaf varie-gation can occur when cells of green and pigment-deficientsectors have distinct genotypes. Cells in green sectors havea wild-type genotypewith functional chloroplasts, while pigment-deficient sectors consist of mutant cells with abnormal plastids.Multiple mechanisms, such as somatic chimerism, transposableelement activity, andorganellar genomemutations, are involved ingenerating cells of a single plant with distinct genotypes (Yu et al.,2007). In other cases, variegation occurs also in mutants withidentical genotype in the green and the chlorotic tissue sectors.Especially the latter cases provide interesting genetic systems forinvestigations into the poorly understood pathways of chloroplastbiogenesis (Sakamoto, 2003; Putarjunan et al., 2013).

Because of the differences in leaf organization between mono-cots and dicots, variegation in monocots is also referred to asstriping. The iojap and albostriansmutants ofmaize (Zeamays) andbarley, respectively, are two well-studied examples in which mu-tations cause a striping phenotype. They belong to the group ofmutants with identical genotype in green and pigment-deficienttissues.The iojapgenecodes for acomponentof the50Ssubunitofthe plastid ribosome. Its role in ribosome assembly or function is

yet unknown (Han et al., 1992; Wanschers et al., 2012). The al-bostrians mutant served as a model for studies on the transcrip-tional machinery of the chloroplast genome (Hess et al., 1993;Zhelyazkova et al., 2012) and on regulatory interactions betweenplastids,mitochondria, and thenucleus (Bradbeeretal., 1979;Hesset al., 1994; Hedtke et al., 1999; Nott et al., 2006). However, elu-cidation of the mechanism leading to the albostrians-specificphenotype of variegation was impeded by the fact that thecausal underlying gene was unknown. The albostriansmutant wasselected in the 1950s after x-ray irradiation of the two-rowed springbarley cv ‘Haisa’. The phenotype is controlled by a single reces-sive nuclear gene that, however, has incomplete penetrance; theprogenyof the fullygreenmutantsegregates intogreen, variegated,and albino seedlings in a ratio of ;1:8:1 (Hagemann and Scholz,1962). Green and variegated seedlings with sufficient photosyn-theticallyactivegreentissuewillgrowtomaturity, remaingreenand/or striped, respectively, and produce fertile flowers. The progenyagainwill consist ofgreen,white, andvariegatedplants (Hagemannand Scholz, 1962). The phenotype is very stable since changes inlight conditions or temperature have remained without effect(HagemannandScholz, 1962;Börneretal., 1976;Hessetal., 1993).Similar to iojap (Walbot and Coe, 1979), the barley ALBOSTRIANS(HvAST) allele (termedHvas inHagemann and Scholz, 1962)wasoriginally described as a plastome mutator, that is, as a nucleargene that induces plastome mutations because the nucleargene–induced albino phenotype showed a stable maternal in-heritance (Hagemann and Scholz, 1962). This hypothesis wasunderpinned originally by the finding of so-called mixed cellscontaining undifferentiated plastids and green chloroplasts(Knoth and Hagemann, 1977). Later, it was proposed, however,that neither iojap nor HvAST is a mutator gene since in eithermutant the albinotic leaf tissues were characterized by

Genetic Control of albostrians Variegation 1431

ribosome-deficient plastids (Börner et al., 1976; Walbot and Coe,1979; Börner andHess, 1993) and the lack of plastid ribosomes isstably inherited like a genuine plastomemutation (Zubko andDay,1998). Indeed, restriction patterns and sequence of the DNA inundifferentiated, ribosome-free plastids of albostrians wereidentical with those of the chloroplast DNA in green tissues ofalbostrians and in the barley wild-type cv ‘Haisa’ and ‘Morex’(Hess et al., 1993; Zhelyazkova et al., 2012). Thus, the barleyHvAST gene product is not expected to act on chloroplast DNAbut to be required for plastid ribosome assembly, maintenance,and/or function.

Recently, ample genomic resources have been developed inbarley (Schulte et al., 2009;Mayer et al., 2011; International BarleyGenome Sequencing Consortium (IBSC), 2012; Ariyadasa et al.,2014;Mascheret al., 2017) andaregreatly facilitatinggenecloning(Mascher et al., 2014). Using these resources, we identifieda candidate gene by positional cloning and confirmed its identitywith ALBOSTRIANS by screening of a barley Targeting InducedLocal Lesions in Genomes (TILLING) population (Gottwald et al.,2009) and by site-directed mutagenesis using RNA-guidedclustered regularly interspaced short palindromic repeats-associated protein9 (Cas9) endonuclease (Jinek et al., 2012).The gene HvAST underlying the albostrians phenotype of varie-gation corresponds to the previously predicted gene HvCMF7,a member of the CCT (CONSTANS, CO-like, and TOC1) motiffamily (CMF) of putative transcription factors (Cockram et al.,2012). The transient analysis of an HvCMF7:green fluorescentprotein (GFP) fusion in conjunction with organelle markers re-vealed a strong subcellular localization of ALBOSTRIANS to thechloroplast.

RESULTS

Map-Based Cloning of the HvAST Gene

The mutant Hvast allele of barley causes variegation, character-ized by green-white striped leaves (Figure 1A). The white leafsectors contain undifferentiated plastids virtually free of 70S ri-bosomes (Hess et al., 1993), while green parts harbor normal,photosynthetically active chloroplasts. We mapped the geneHvAST to the long arm of chromosome 7H using two F2mappingpopulations (Morex 3 M4205 [MM4205] and Barke 3 M4205[BM4205]) each representing 182 gametes. Further fine mappingof the gene in 2688 gametes of population MM4205 delimited theHvAST target region to a 0.06 centiMorgans interval (Figure 1B).The closest HvAST flanking markers were anchored to thephysical map of barley (International Barley Genome SequencingConsortium (IBSC), 2012; Mascher et al., 2013) and by sequencecomparison and BAC library screening, five overlapping physicalmapcontigs (Finger printed contigs) spanning adistanceof;0.40mega base pair (Mbp; between flanking markers Zip_2661 and3_0168) were identified. Next, 60 BAC clones representing thephysical target interval were sequenced, providing novel in-formation for developing andmappingof newBACcontig-derivedmarkers, which delimited the HvAST locus to the two overlap-ping BAC clones HVVMRXALLrA0395M21 and HVVMRXALL-mA0230A06 (Figure 1C). Eventually, a 46-kilobasepair regionwasfound to be the smallest genetic and physical interval containing

theHvAST locus (Figure 1D). Sequence comparison to annotatedgenes defined on the ‘Morex’ draft whole genome shotgun as-sembly (International Barley Genome Sequencing Consortium(IBSC),2012) revealed thepresenceofasingle ’Morex’gene locus,MLOC_670 (GenBankaccession numberAK366098).MLOC_670was previously named HvCMF7 in a study on the evolution of theCMF in Poaceae (Cockram et al., 2012). The HvCMF7 genestructure, supported by cDNA analysis, consists of three insteadof two exons as in silico predicted forMLOC_670. We discovereda 4-bp deletion by resequencing of the HvCMF7 gene in the al-bostrians mutant (line M4205) compared with the wild-type ge-notype (Figure 1E). This mutation is predicted to induce a shift ofreading frame and, as a consequence, a premature stop codon inthe second exon of the gene. This lesion is expected to result in(partial) loss of the gene function in the mutant and makes theHvCMF7 gene locus a very strong candidate for representing theHvAST gene. Themutant allele in the original albostriansmutant isdesignated as Hvcmf7-1.

Functional Validation of the HvCMF7 Gene by TILLING andTest of Allelism

Toverify thebiological functionof thegeneHvCMF7,wescreenedanethylmethanesulfonate (EMS)–inducedTILLINGpopulation (cv‘Barke’; Gottwald et al., 2009) for independent mutated alleles.Forty-twoEMS-inducedmutations, including20synonymousand20 nonsynonymous mutations, one 9-bp deletion, and one mu-tation leading toaprematurestopcodon,were identified.Whereasno other mutant family exhibited any chlorophyll/photosynthesis-related phenotype, the M3 progeny of the M2-TILLING family6460-1 (carrying the premature stop codon) segregated for al-binism.All homozygousmutant progenyof TILLING family 6460-1showed a complete albino phenotype (Figures 2A and 2B), whilethe homozygous wild-type and heterozygous plants from thesegregating M3 population all had entirely green seedlings. Thelinkage between albino phenotype and mutant genotype of theHvCMF7 candidategenewas further validated throughanalysis ofa largeM4populationcomprising245 individuals derived fromfiveM3 heterozygous plants (Figure 2A). All homozygousM4mutantsgrew into purely albino seedlings. The Mendelian segregationpattern (x250.74;df52; P50.69) of theM4generation indicatedthat, like the albostrians phenotype in M4205, the albino pheno-type was controlled by a single recessive gene. We observedexceptions from the pure albino phenotype in homozygous mu-tantM4 andM5 progenies of TILLING family 6460-1 in the case oftwo seedlings showing very narrow green sectors on the first leaf(Figures 2C), indicating an analogous, yet much more severephenotype compared with the variegation conferred by theoriginalHvCMF7mutation (i.e., the 4-bp deletion). Because of theseverity and predominance of albino sectors, both M4 and M5striped plants did not develop beyond the seedling stage(Figure 2D). The pre-stop allele in the identified TILLING family6460-1 is designated as Hvcmf7-2.The prominent characteristic of the albostrians mutant is the

lackof 70S ribosomes in theplastidsof albinotic leaf sectors (Hesset al., 1993). We checked therefore whether in albino leaves of theallelic TILLINGmutant 6460-1 plastid ribosomes are alsomissing.Indeed, as in albostrians, ribosomes could not be detected by

1432 The Plant Cell

Figure 1. Map-Based Cloning of the HvAST Gene.

(A) Examples of variegation of leaf coloration of the homozygous albostrians mutant plants.(B)High-resolution geneticmapping of theHvAST gene. One cosegregatingmarker (Zip_2662) was identified, and the recombination between contiguousmarkers is indicated by the numbers below. 7HL, long arm of barley chromosome 7H.(C)Physical anchoring ofmarkers to the sequencedMTPBACs. The two flankingmarkers Zip_2661andContig_92279 aswell as the cosegregatingmarkerZip_2662were located on the sameBAC clone, HVVMRXALLrA0395M21 (FP contig_7112), as indicated in green color. Each gray bar represents one BACclone. FP contig, Finger printed contig, defined as a set of overlapping BAC clones.(D)Gene prediction based on repeatmaskedBAC assemblies of the target interval through alignment to barley genemodels published by the InternationalBarley Genome Sequencing Consortium (IBSC) (2012). A single gene,MLOC_670 (GenBank accession number AK366098) as indicated by the arrow, wasidentified within the target interval between the two flanking markers Zip_2661 and Contig_92279, flanking a physical distance of ;46 kilobase pairs.(E)Structure of the geneHvAST. Themutant allele harbors a 4-bp deletion (red bar) near the end of the first exon comparedwith thewild-type allele found ingenotype ’Morex’. The gene structure of HvAST is according to cDNA analysis of barley cv ’Morex’. ORF, open reading frame; UTR, untranslated region.

Genetic Control of albostrians Variegation 1433

electron microscopy in defective plastids of the TILLING mutant(Figure 3A), and the plastid rRNAs were missing in preparationsfrom albino leaves as determined by formaldehyde agarose gelelectrophoresis and evaluation using an Agilent 2100 Bioanalyzer(Figure 3B). Consequently, the development of plastids in thealbino sectors of the TILLING mutant was impeded to a similarextent as in the albostrians defective plastids. In contrast to thewell-developedcrescent-shapedchloroplasts in thewild typeandgreen TILLING mutants, smaller and irregularly shaped plastidswere observed in the albostrians and albino TILLING mutants.Instead of typical thylakoids and granum stacks as seen in thewild-type chloroplasts, only a few vesicle-like structures werefound within mutant plastids (Figure 3A).

Crosses between plants carrying the original 4-bp deletionHvCMF7 allele of genotypeM4205 (Hvcmf7-1/Hvcmf7-1) and thenovel TILLING pre-stop allele (HvCMF7/Hvcmf7-2) demonstrated

the allelic state of both mutations. Seven of the 18 F1 plantsshowed a green phenotype as expected for plants heterozygousfor theoriginalHvCMF7allele (HvCMF7/Hvcmf7-1). The remaining11 plants (Hvcmf7-1/Hvcmf7-2), heterozygous for the two mu-tated alleles, were either completely albino or green-white var-iegated (Figures 2E and 2F). This analysis confirmed thatHvCMF7is the functional gene underlying the mutant phenotype of theoriginal albostrians mutant genotype M4205.

Site-Directed Mutagenesis of HvCMF7 by RNA-GuidedCas9 Endonuclease

That HvCMF7 was the causal gene for the striped/albino phe-notype was confirmed by the allelism of the albostrians andTILLINGmutants. Remarkably, thephenotypeof the homozygousTILLING mutant was much more severe than that of the original

Figure 2. Functional Validation of HvCMF7 by TILLING and Allelism Test.

(A) Phenotypic segregation population in M4 progeny of TILLING family 6460-1. Yellow arrows indicate homozygous pre-stop TILLING mutants, whichshow an albino phenotype and are delayed in growth compared with the wild type. The plants shown here are derived from three heterozygous M3 plants(i.e., 6460-1_4, 6460-1_9, and 6460-1_11).(B) Example of homozygous HvCMF7 pre-stop TILLING mutant (Hvcmf7-2), which exhibits the albino phenotype.(C) Examples of striped phenotype of the TILLINGmutants. A single stripedmutant was identified in bothM4 generation (6460-1_9_19) andM5 generation(6460-1_4_21_19).(D) Phenotype of the striped M5 plant at later developmental stage. Neither variegated mutants reached the reproductive stage.(E) and (F) F1 hybrids of original albostriansmutant (Hvcmf7-1) and pre-stop TILLINGmutant (Hvcmf7-2) show either albino (MZ35-5_4) (E) or green-whitestriped (MZ35-11_2) (F) phenotype.

1434 The Plant Cell

albostrians mutant, which we hypothesized could be due to thedifferent locations of the two mutations within the coding region(Figure 4). In an attempt to test this hypothesis and to reproducethe albostrians phenotype, we used site-directedmutagenesis byRNA-guided Cas9 endonuclease to generate new HvCMF7 mu-tations close to the original site of the albostrians mutation ofM4205. Two HvCMF7-specific guide RNAs (gRNAs) surrounding

the 4-bp deletion region of the original albostrians mutant wereselected (Figures 5B and 5C).Twenty-one of 23 T0 regenerated plantlets carried the intact

T-DNA, that is, an OsU3 promoter-driven gRNA and a maizecodon-optimized Cas9 controlled by the maize Ubiquitin1 pro-moter with first intron. Twenty of these showed fully green leaves,whereas one plant (BG684E11, carrying the gRNA specific for

Figure 3. Ultrastructural Analysis Of The Hvcmf7-1 And Hvcmf7-2 Mutants And Analysis Of rRNA From The Wild Type And Hvcmf7-2 Mutant Using AnAgilent 2100 Bioanalyzer.

(A) Ultrastructural analysis of the Hvcmf7-1 and Hvcmf7-2mutants. Green leaves of the Hvcmf7-1mutant (panels 1 and 5) and the wild type (for HvCMF7gene locus) TILLING plant (panels 3 and 7) contain chloroplasts with fully differentiated grana and stroma thylakoids. By contrast, only some vesicle-likestructures andplastoglobuli were observed in plastids of albino leaves of theHvcmf7-1 (panels 2 and6) andHvcmf7-2 (panels 4 and8)mutants. The bottompanels represent largermagnificationof thecorrespondingplastid in the toppanels.Bar inpanels 1 to451mm;bar inpanels 5 to85200nm.GA,grana;PL,plastoglobuli; ST, stroma thylakoid; VL, vesicle-like structure.(B)Analysis of rRNA from thewild type andHvcmf7-2mutant using an Agilent 2100 Bioanalyzer. Homozygouswild type (a), green sectors of theHvcmf7-2mutant (b), andwhite sectorsof theHvcmf7-2mutant (c). Separation of totalRNAextracted from thesamplesmentionedabove in formaldehydeagarosegel(d). FU, fluorescence unit.

Genetic Control of albostrians Variegation 1435

target motif 1, which resides a few base pairs upstream of theoriginal albostriansmutation site) exhibited a green-white stripedphenotype resembling that of mutant M4205. A variegated leaf ofBG684E11 was used for genotyping the preselected HvCMF7-region, which revealed two mutant alleles each carrying a singlebase-pair (A/T) insertion within target motif 1 at the position ex-pected to be cut by the gRNA/Cas9 complex (Figure 5D).

Genotyping revealed the chimeric nature of the original T0 plantBG684E11, since four genotypic classes could be observed in thecorresponding T1 progeny: homozygous mutant 1/mutant 1 ormutant2/mutant2 (class I), heterozygousmutant1/mutant2 (classII),mutant 1 ormutant 2/wild type (class III), and homozygouswildtype/wild type (class IV; Figure 5E). The individuals of class I andclass II either showed green-white variegated (Figure 5A) or albinoleaves, while plants in the remaining two classes exclusively werecompletely green. Hence, site-directed mutagenesis of HvCMF7by RNA-guided Cas9 endonuclease reproduced a phenotypesimilar to albostrians, which unambiguously confirmed HvCMF7as the functional associatedgene.Ourfindingsalso supported thehypothesis that the severity of the HvCMF7 mutant phenotypedepended on the relative position of lesions within the gene. TheCas9-inducedmutant allelewith 1-bp insertion (nucleotideG)wasdesignated as Hvcmf7-3.

HvCMF7 Is a Member of the CMF Genes and Is Targeted tothe Plastids

Sequence comparison revealed thatHvCMF7 encodes a putativeproteinwith a lengthof 459aminoacids. It carries aCCTdomain ataminoacidposition403 to447according tosurveyon theNationalCenter for Biotechnology Information’s Conserved Domain Da-tabase (Marchler-Bauer et al., 2017). CCT is a conserved domain

of 43 amino acids found in the Arabidopsis (Arabidopsis thaliana)transcription factors CONSTANS (CO), CO-LIKE, and TIMINGOFCHLOROPHYLL A/B BINDING PROTEIN1 (TOC1; Putterill et al.,1995; Strayer et al., 2000). Based on public gene expression dataderived fromeight tissues/growthstagesofbarley, asdescribed inInternational Barley Genome Sequencing Consortium (IBSC)(2012), the geneHvCMF7 is expressed in all eight tissues/stages,reaching the highest level of expression in young barley tillers andthe lowest expression in the embryo and developing grains, re-spectively (Supplemental Figure). CMF proteins were pro-posed to represent transcription factors (Ben-Naim et al., 2006),and the CCT domain was predicted to be a nuclear localizationsignal (Robert et al., 1998). For HvCMF7, in silico prediction(Emanuelsson et al., 1999, 2000; Small et al., 2004) indicated athigh probability the presence of an N-terminal chloroplast transitpeptide (cTP), suggesting chloroplast targeting of the protein. Wetested the subcellular localization after fusing GFP to the C ter-minus of HvCMF7.Four constructs were made for HvCMF7: wild-type HvCMF7

(HvCMF7:GFP), two mutated forms (i.e., the original albostriansmutant M4205 [Hvcmf7-1:GFP] and the TILLING mutant 6460-1[Hvcmf7-2:GFP]), and the cTP-encoding domain of HvCMF7(cTP_83AA_HvCMF7:GFP; Figure 6A). These fusion constructs,together with the plastid marker pt-rk-CD3-999 (Nelson et al.,2007), respectively, were subjected to transient expression ex-periments using biolistic bombardment of barley leaf segmentswith vector-coated gold particles. The plastid marker pt-rk-CD3-999 (Nelson et al., 2007) was detected by its orange fluorescence(Figure 6, mCherry column). Cells expressing only the GFP(nonfusion GFP control) showed green fluorescence associatedwith the nucleus and the cytoplasm, but not with plastids(Figure 6B). By contrast, the mCherry signal of the plastid marker

Figure 4. Alignment of HvCMF7 Protein Sequences Encoded by the Wild-Type and Mutant Alleles.

Thepositionof thestopcodonof the truncatedproteins is indicatedwithanasterisk.Thealteredaminoacidsof the truncatedproteinsare indicatedbypurpleunderlines. The CCT domain of HvCMF7 protein is indicated by green underline. Alignment was generated with the online tool Clustal Omega and all gapswere deleted. The figure was prepared by visualization with the online Jalview applet.

1436 The Plant Cell

accumulated exclusively in plastids (Figure 6C). HvCMF7:GFPand its two truncated mutant forms were detected as greenfluorescence in leaf epidermal cells associated with the nucleiand more strongly with the plastids (Figures 6D to 6F). Theplastid localization of HvCMF7 was further confirmed by thecTP_83AA_HvCMF7:GFP construct, which produced strongaccumulation ofGFPsignals in theplastids (Figure 6G). In total, 30

to 50 cells for each of the five fusion constructs were checked forthe presence of both green and orange fluorescence.To furtherconfirmthesubcellular localizationofHvCMF7,stably

transgenic barley lines carrying HvCMF7:GFP were obtainedthrough Agrobacterium-mediated transformation. Consistentwith results of the transient experiments, GFP fluorescencewas specifically targeted to the plastids in the epidermal cells

Figure 5. Site-Directed Mutagenesis of HvCMF7 Gene by RNA-Guided Cas9 Endonuclease.

(A) Examples of the variegated homozygous HvCMF7 mutants.(B) Gene structure of the HvCMF7 gene. The region of the 4-bp deletion carried by the original albostrians mutant is marked with a red bar.(C) The genomic target motifs 1 and 2 (underlined) were selected up- and downstream of the 4-bp deletion (indicated by green letters) in the originalalbostriansmutant. Guide RNAs were designed to address the sequences indicated by gray background. The protospacer adjacent motifs bound by theCas9 protein are marked with blue color.(D)Mutation detection in T0 plants. One biallelic mutant was identified that carries a one-nucleotide insertion 3 bp upstream of the protospacer adjacentmotif of target motif 1.(E) Inheritanceofmutations in theT1generation.Theprogeniesof theT0homogeneouslybiallelicmutantsegregated into four classesofplantsbasedon thegenotypic status of the HvCMF7 gene. All the homozygous (class I) and biallelic mutants (class II) exhibit either green-white striped or albino phenotypes,while heterozygous/chimeric mutants with wild-type allele (class III) and homozygous wild type plants show the green phenotype.

Genetic Control of albostrians Variegation 1437

Figure 6. Subcellular Localization of HvCMF7:GFP Fusion Proteins.

(A)Schematic drawingof theconstructs used inbiolistic bombardment. cTP_83AA_HvCMF7,N-terminal cTPofHvCMF7with a lengthof 83aminoacids aspredicted byonline tool PredSL (Petsalaki et al., 2006); HvCMF7, coding sequenceofwild-typeHvCMF7gene;Hvcmf7-1, coding sequenceofmutant alleleHvcmf7-1 of the original albostriansmutant lineM4205;Hvcmf7-2, coding sequence ofmutant alleleHvcmf7-2of the pre-stop TILLINGmutant line 6460-1;mCherry, mCherry fluorescent protein; pCaMVd35S, cauliflower mosaic virus double 35S promoter; pZmUbi, maize Ubiquitin1 promoter; tNOS, Agro-bacterium NOPALINE SYNTHASE terminator. The schematic drawing is not in proportion with gene length.(B) Bombardment using gold particles coated with pSB179 plasmid (expressing GFP driven by the maize Ubiquitin1 promoter) as control. The greenfluorescence is observed in the nucleus and the cytoskeleton/cytoplasm.(C) Biolistic assay for plastid marker pt-rk-CD3-999 that carries the mCherry gene driven by a double 35S promoter. The mCherry signal (orange color)exclusively accumulates in the plastids.(D) Particle cobombardment of both plastid marker pt-rk-CD3-999 and the wild-type fusion construct HvCMF7:GFP. The bright-field, GFP, and mCherrysignals are displayed as individual channels, and the merged channels are shown on the rightmost panel. GFP and mCherry fluoresce green and orange,respectively.

1438 The Plant Cell

(Figure 6H). Surprisingly, we could not detectGFP fluorescence inthe mesophyll cells.

Taken together, the wild-type HvCMF7 and its two mutatedforms displayed a clear compartmentalized accumulation in theplastids. Although nuclear fluorescence was also visible in thetransformed cells, which would support a role of the CCT domainas a nuclear localization signal, further investigations are requiredto discriminate whether the observed localization of HvCMF7 tothe nucleus is only the consequence of unspecific nuclear tar-geting of GFP aswas reported in other systems previously (Seibelet al., 2007).

DISCUSSION

Leaf variegation in the barley mutant albostrians is distinct due tothe incomplete penetrance of the phenotype. Even when carryingthe mutant allele at homozygous state, plants may be completelygreenand theprogenyofsuchagreenplantwill segregate ina ratioof 1:8:1 for green, variegated, or albino phenotype, respectively.This phenomenon implies residual activity of the affected alleleand a threshold mechanism involved at a critical decision point inchloroplast development/maturation—a hypothesis that can betested now that we have isolated theHvCMF7 gene by positionalcloning.

Our successful cloning ofHvCMF7 is supported by four lines ofevidence: (1) the smallest genetic interval contained only a singlegene in perfect linkage with the phenotype, (2) mutant analysis byTILLING revealed an independent pre-stop allele leading to a re-lated but more severe and fully penetrant phenotype, (3) crossingof the two independent mutant alleles demonstrated their state ofallelism, and (4) albostrians-like alleles were induced by site-directed mutagenesis using RNA-guided Cas9 endonuclease.In the original albostrians mutant allele, Hvcmf7-1, with the ge-notype M4205, a 4-bp deletion at position 1123 to 1126 of thecoding sequence is predicted to induce a shift of reading frameand, as a consequence, a premature stop codon in the secondexon of the gene (Figure 4).

During functional validation of the identified gene, the char-acterization of two novel independent mutant alleles, Hvcmf7-2and Hvcmf7-3, implied that severity and penetrance of thephenotype are correlated with the relative position of the mu-tation as well as with allele dosage. The albino phenotypeconferred by Hvcmf7-2, induced by EMS and identified byTILLING, suggested (almost) complete loss of protein activity.Although this phenotype is not identical with the mostly stripedprogeny of albostrians barley, Hvcmf7-2 provided evidence forthe identified geneHvCMF7 representing albostrians, since also

two albino seedlings with tiny green stripes could be observedamong 289 M3, M4, and M5 progenies. F1 plants heterozygousfor Hvcmf7-1/Hvcmf7-2 confirmed their allelism but also dem-onstrated that the phenotype is affected by allele dosage. Incontrast to M4205 (Hvcmf7-1/Hvcmf7-1), F1 plants (Hvcmf7-1/Hvcmf7-2) were either albino or green-white variegated, but nota single fully green F1 could be observed. Since plants het-erozygous with one wild-type allele (HvCMF7/Hvcmf7-1) aregenerally fully green, the severity of phenotype thus varies in anallele dosage–dependent manner.The phenotypic difference of Hvcmf7-1 and Hvcmf7-2 homo-

zygous mutants provided the first indication of a correlation be-tween relative position of the mutation and severity of thephenotype. This hypothesis was confirmed by a third allele,Hvcmf7-3, obtained via site-directed mutagenesis using RNA-guided Cas9 endonuclease. A one-nucleotide insertion at 28 bpupstream of the 4-bp deletion inHvcmf7-1 led to a frameshift andaHvcmf7-1-similar gene product of 10 amino acids longer alteredputative C-terminal sequence. Homozygous seedlings carryingthis mutation (Hvcmf7-3/Hvcmf7-3) resembled the typical al-bostrians green-white striped or albino seedling phenotype;however, in contrast to Hvcmf7-1/Hvcmf7-1 plants, no greenseedlings were observed, indicating amore severe phenotype forHvcmf7-3/Hvcmf7-3 plants.

HvCMF7 Is a CCT Domain Protein and Homolog of theArabidopsis CIA2 Protein

In silico analysesof the amino acid sequence ofHvCMF7 revealeda putative cTP at its N terminus and a CCT domain near theC terminus, qualifying it to be a member of the larger CCTdomain–containing gene family of the Poaceae (Cockram et al.,2012). The CCT domain is named after rather well-studied planttranscription factors: CO, CO-LIKE, and TOC1 (Strayer et al.,2000).HvCMF7 belongs to a subgene family that is characteristicfor encoding only a single CCT domain per gene (Cockram et al.,2012), and CCT is the only domain of HvCMF7 in common withthese transcription factors.The closest homologs of HvCMF7 in Arabidopsis are the

genes CHLOROPLAST IMPORT APPARATUS2 (AtCIA2) andCHLOROPLAST IMPORT APPARATUS 2-LIKE (Sun et al., 2001).AtCIA2 encodes a nucleus-localized transcription factor involvedin expression of both protein translocon and ribosomal proteingenes by binding to the respective promoters (Sun et al., 2009a).Yet, HvCMF7 in barley might have a different function. As ex-pected by the in silico–predicted cTP, HvCMF7 localized to barleychloroplasts in transient colocalization and stable transformation

Figure 6. (continued).

(E) and (F)Subcellular localization of the twomutant proteins Hvcmf7-1:GFP (E) and Hvcmf7-2:GFP (F)was also investigated by cobombardment with theplastid marker pt-rk-CD3-999 and display the same subcellular localization as the wild-type protein (D).(G) Particle cobombardment of both plastid marker pt-rk-CD3-999 and fusion construct cTP_83AA_HvCMF7:GFP.(H)Subcellular localization of HvCMF7:GFP in barley stable transgenic lines. TheGFP fluorescence, characterized by photospectrometric analysis with anemission peak at wavelength 509 nm, was specifically accumulated in the plastids.Theyellowarrows in themergedpanels indicate thenucleus.Thefirst leaf of10-d-oldbarley seedlingswasused forparticlebombardment. Thefluorescencewas checked 24 h after bombardment. Bar for all images 5 20 mm.

Genetic Control of albostrians Variegation 1439

experiments. GFP signal was also observed in the nucleus; this,however, does not represent evidence of dual targeting to theplastid and nucleus, since GFP has a general tendency of nuclearlocalization as demonstrated by the GFP-only controls in thetransient expression experiments. Searching the public plantproteomics database (Sun et al., 2009b) revealed no HvCMF7homologs of other plant species targeted to the plastids.HvCMF7is the only CCT domain protein found to be targeted to plastidsto date.

Nevertheless, the exactmolecular function of HvCMF7 remainselusive. Based on the analysis of independent HvCMF7 mutantalleles, it can be concluded that the CCT domain may be involvedin overall HvCMF7 function; however, this domain is not exclu-sively required to regulate proper chloroplast differentiation ormaturation in barley. The allelesHvcmf7-1 andHvcmf7-3 allow, atleast in a threshold-dependent manner, the formation of fullyfunctional chloroplasts in green seedlings and green leaf sectors;hence, normal differentiation of chloroplasts despite completeloss of the CCT domain. Both mutated alleles most likely reducethe protein functionality, but do not destroy its function. The in-creasingly severe phenotype in mutant alleles with defects pro-gressing toward theN terminusof thegene indicated thepresenceof an additional, still-to-be-described domain of the protein that isinvolved in communicating its essential function in the differentcell compartments. Further protein–protein and DNA–protein in-teraction studies involving the series of barley HvCMF7 mutantswill be required to further address this question.

Notably, we found phenotypic effects only for mutations thataffect large portions of the HvCMF7 protein at its C terminus. TheTILLING population contained also several mutations that in-troduced putative synonymous or nonsynonymous exchanges ofamino acids into the protein sequence. All of these showed thewild-type phenotype. The gradual increase in the amount of whiteleaves and white leaf sectors with shifting the position of themutation toward the N terminus of the HvCMF7 protein and theabsence of effects of point mutations leading to an altered aminoacid in this region suggest that the C-terminal part of the proteindoes not have a catalytic function but potentially rather interactswith other proteins, possibly involved in expression or regulationof genes required for plastid ribosome formation. In case of themutant Hvcmf7 alleles, this interaction would be less efficient ormissing, suggesting the formation of a multiprotein complex withless or no remaining activity.

HvCMF7: A Factor Involved in ChloroplastRibosome Formation?

Remarkably, green, green-white striped, and white seedlings ofthe albostrians mutant have all the same genotype; they are ho-mozygous for the recessive mutant allele (Hvcmf7-1) and theyhave identical plastid genomes (Hess et al., 1993; Zhelyazkovaet al., 2012). The different fate of plastids in albostrians leaves istherefore not causedbyamutation in theDNAof thoseproplastidsthat do not develop into chloroplasts. There is no environmentalinfluence on the phenotype of albostrians plants, in contrast toother variegated plants that have identical genotypes in differentlycolored leaf sectors, such as barley tigrina mutants or the Ara-bidopsis immutans mutant (Wettstein et al., 1974; Wetzel et al.,

1994). In these mutants, the pigmentation of leaves and leafsectors depends on the light conditions; hence, illumination in-duces chlorosis due to photooxidation (Wettstein et al., 1974;Wetzel et al., 1994; Lee et al., 2003; Putarjunan et al., 2013). Mostcertainly, it is the ribosome deficiency of all or only part of theplastids, respectively, in the meristems of the first and followingleaves that blocks further chloroplast development and thuseithercauses the formation of completely white or green-white varie-gated leaves, respectively. As was demonstrated previously(Knoth and Hagemann, 1977; Dorne et al., 1982) and confirmed inthis study, plastids in white leave sectors of albostrians plants donot contain ribosomes; thus, HvCMF7 is needed directly or in-directly for the biogenesis of plastid ribosomes. Since neitherHvCMF7 nor its homologs in Arabidopsis have been described asa component of plastid ribosomes, that is, as a ribosomal proteinsensu stricto (Yamaguchi and Subramanian, 2000; Yamaguchiet al., 2000; Tiller et al., 2012), the HvCMF7 protein might not beassociated with ribosomes or be associated only under certainconditions and might be required for the assembly of plastid ri-bosomes rather than for their function.In conclusion, the identification of the barley geneHvCMF7 has

revealed insights into the roleofCCTdomain–containingproteins.Weshow that abarleyCCTdomain–containingprotein is localizedtoplastids. This gene likely plays a crucial role during early embryodevelopment for plastid ribosome formation and hence forchloroplast development. An Arabidopsis homolog of HvCMF7was previously reported to act as transcriptional regulator fornuclear genes coding for chloroplast ribosomal proteins and forthechloroplastprotein translocon (Sunetal., 2009a).Basedonourfindings, adual roleof this special groupofCCTdomainproteins innuclei and in plastids cannot be ruled out. The identification of thegenecausing thealbostriansphenotypewill foster studiesonearlyphases of chloroplast development, in particular the formation ofplastid ribosomes, onpossibledual localizationofCCTproteins tonuclei and plastids, and on leaf variegation in nonchimeric plants.

METHODS

Plant Materials

The six-rowed spring barley cv ‘Morex’ and the two-rowed spring barley(Hordeum vulgare) cv ‘Barke’ were used as maternal parent in crossingswith the mutant line M4205 (Hagemann and Scholz, 1962), respectively.The obtained F1 progeny were self-pollinated to obtain F2 populations forgenetic mapping. The two F2 mapping populations were designated as‘MM4205’ (‘Morex3M4205’) and ‘BM4205’ (‘Barke3M4205’), indicatingthe respective parental combinations of the crosses. All plantswere grownunder greenhouse conditions with a day/night temperature and photo-period cycle of 20°C/15°C and 16-h light/8-h darkness, respectively.Supplemental light (at 300mmol photons m22 s21) was used to extend thenatural light with incandescent lamps (SON-T Agro 400, MASSIVE-GROW).

DNA Preparation

DNAwas isolated according to the protocol ofDoyle andDoyle (1990) fromleaf samples collected from three-leaf stage, greenhouse-grown seedlingsand quantified using a NanoDrop spectrophotometer (Thermo FisherScientific) according to the manufacturer’s instructions and adjusted to100 ng/mL for any PCR application.

1440 The Plant Cell

PCR

DNA amplification reactions were performed in a total volume of 20 mLcontaining40ngof templateDNA, 4mMdNTPs, 1mLeachof 5mMforwardand reverse primers, 0.5 units of HotStarTaq DNA polymerase (Qiagen),and2mLof 103PCRbuffer (100mMTris-HCl, pH8.3, 500mMKCl, 15mMMgCl2, and 0.01% gelatin). Touch-down PCR program was used witha GeneAmp 9700 thermal cycler (Life Technologies): initial denaturation at94°C for 15 min followed by five cycles at 94°C for 30 s, annealing at 65 to60°C (21°C/cycle) for30s,extension1minat72°C,and thenproceeded for40 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 1 min, and followed byafinal extensionat72°C for10min.Detailed informationof theprimersusedfor marker development is presented in Supplemental Table 1.

CAPS Assay

Single-nucleotide polymorphisms (SNPs) identified between the mappingparents were converted into cleaved amplified polymorphic sequences(CAPS)markers (Supplemental Table 1; Thiel et al., 2004). For this purpose,PCR products were purified using the NucleoFast 96 PCR kit (Macherey-Nagel) and sequenced on an ABI 3730 XL DNA analyzer (Life Technolo-gies). SequenceswerealignedbyusingSequencher version5.2.3 software(Gene Codes, https://www.arabidopsis.org/servlets/TairObject?type5stock&id53001623338) for SNP identification. Subsequently, SNP2CAPSsoftware (Thiel et al., 2004) was adopted to select a suitable restrictionendonuclease. The resulting fragments were resolved by electrophoresison 1.5% (w/v) agarose, 13 Tris Base Borate Ethylenediaminetetraaceticacid (EDTA) gels (Invitrogen).

Genetic Mapping

As an initial step, 91 F2 individuals from each population ‘MM4205’ and‘BM4205’were selected for low-resolution geneticmapping of theHvAST/HvCMF7 gene. Genotyping was performed by using the Illumina GoldenGate assay with a custom set of 381 BOPA SNP markers (Close et al.,2009). Subsequently, only the ‘MM4205’ population was further used forfine mapping of HvCMF7 due to its higher rate of polymorphic markerscompared with the ‘BM4205’ population. Saturation mapping within the‘MM4205’ population (91 genotypes) was conducted in an effort to narrowdown the target genetic interval. Next, fine mapping of HvCMF7 with anadditional 1344 F2 plants was scheduled in two steps. First, 142 re-combinants were selected by screening 960 F2 plants with flankingmarkers CAPS_2536 and CAPS_2560 and used for further marker satu-ration. In a second step, 384 variegated or albino F2 individuals wereselected from 1920 F2 plants and screened for recombination betweennewly identified flanking markers Zip_2661 and Zip_2680_1. In addition,the genotype at theHvCMF7 locus was determined by phenotyping 30 F3seedlings derived from each F2 recombinant through self-pollination.While wild-type F2 plants are expected to yield 100% green progeny, F3families derived from heterozygous F2 will segregate into 75% wild-typeand 25%variegated or albino plants. F3 progeny of F2 homozygous greenmutants will segregate into 10% green, 80% variegated, and 10% albinoseedlings. All molecularmarkers used for saturationmapping aswell as forfine mapping relied on publicly available genomic resources (Sato et al.,2009; Mayer et al., 2011; International Barley Genome Sequencing Con-sortium (IBSC), 2012).

Linkage Analysis

Genetic linkage analysis was performed using JoinMap 4 software (VanOoijen, 2006). Homozygous wild-type, heterozygous, and homozygousmutant allele calls were defined as A, H, and B, respectively; missing datawere indicated by dashes. Maximum likelihood algorithm and Kosambi’smapping functionwerechosen for building the linkagemaps.Markerswere

assigned intosevengroupsbasedon logarithmofodds (logarithmofodds54)groupings. Visualization of maps derived from JoinMap 4 was achieved byMapChart software (Voorrips, 2002).

Physical Mapping

The target physical region was identified by anchoring the flanking geneticmarkers to the physical map of barley (International Barley Genome Se-quencing Consortium (IBSC), 2012). Two anchoring strategies were ap-plied: (1) in silico anchoring approach through BLASTn (Mount, 2007), thatis, sequenceof themarkerswereused toscreenbyBLASTagainst thedraftbarley sequence assembly at Leibniz Institute of Plant Genetics and CropPlant Research (IPK) barley BLAST Server (http://webblast.ipk-gatersleben.de/barley/) or the HarvEST Server (http://138.23.178.42/blast/index.html); (2) experimental anchoring via PCR screening of a BAClibrary derived from barley cv ‘Morex’ (Schulte et al., 2011) as describedpreviously (Supplemental Table 2; Ariyadasa and Stein, 2012). Theminimaltilingpath (MTP)BACsspanning theregion representedby thephysicalmapcontigs were shotgun sequenced (Beier et al., 2016) on the MiSeq System(IlluminaMiSeq;SupplementalTable3).Theshotgun readswereassembledas described previously (International Barley Genome Sequencing Con-sortium (IBSC), 2012). Gene models were predicted on nonrepetitive se-quences (Schmutzer et al., 2014) of the target interval through alignment ofgene models defined on the Morex WGS assembly (International BarleyGenome Sequencing Consortium (IBSC), 2012).

TILLING Screening

AnEMS-induced TILLINGpopulation, comprising 7979M2plants, derivedfrom a two-rowed malting barley cv ‘Barke’ (Gottwald et al., 2009), wasused for identificationof independentmutated alleles of theHvCMF7gene.Primers were designed covering the coding sequence of HvCMF7(Supplemental Table 4). PCR amplicons were analyzed combined withdouble-stranded DNA cleavage kit (DNF-480-3000) and Gel-double-strandedDNAreagentkit (DNF-910-K1000) according to themanufacturer’sprotocols (Advanced Analytical Technologies GmbH). Subsequently, thecleaved PCR products were separated using the AdvanCE FS96 capillaryelectrophoresis system (Advanced Analytical Technologies), and resultswere interpreted with assistance of the PRO Size software (AdvancedAnalytical Technologies). The identified M2 TILLING mutants were con-firmed by Sanger sequencing of PCR amplicons derived from the respec-tive families. Plant families carrying nonsynonymous mutations, deletions,or immature stop codons were selected for propagation. Phenotypingwas performed throughM3 to M5 generation of the identified M2 mutants.Heterozygous plants were propagated and maintained for further re-production. All the identified TILLING families carrying mutation for theHvCMF7 gene are summarized in Supplemental Table 5.

HvCMF7 Gene Structure Analysis

Primary leaf tissuewascollected fromseedlings4dafter germination. TotalRNA was extracted using TRIzol reagent (Invitrogen) following manu-facturer’s instructions. The concentration of the obtained RNA was de-terminedbyhelpof aQubit 2.0fluorometer (LifeTechnologies) according tomanufacturer’s manual. Genomic DNA was removed from RNA prepa-rations by incubation with RNase-free DNase I (Fermentas) following themanufacturer’s instructions. The reactions were performed in a total vol-ume of 10 mL containing 1 mg of RNA, 1 mL of 103 reaction buffer withMgCl2 (100mMTris-HCl, pH7.5, 25mMMgCl2, and1mMCaCl2) and1unitofDNase I (1U/mL).Sampleswere incubatedat37°C for30min, followedbyadding 1mL of 50mMEDTA and further incubation for 10min at 65°C. TheDNase I–treated RNA was then used as template for cDNA synthesis. RTwas performed using the SuperScript III First-Strand Synthesis SuperMix

Genetic Control of albostrians Variegation 1441

for quantitativeRT-PCR (Invitrogen) following themanufacturer’sprotocol.The DNase I–treated RNAwas used as template in a total volume of 30 mLcontaining 15 mL of 23 RT reaction mix, 3 mL of RT enzyme, and 2 mL ofdiethylpyrocarbonate (DEPC)–treatedwater. RT reactionswere performedduring the following cycling profile: 25°C for 10 min, 50°C for 30min, 85°Cfor 5 min, with a final hold at 4°C. Subsequently, the RNA strand wasremoved fromobtained cDNAby incubating at 37°C for 20min after adding1mL ofEscherichia coliRNaseH. PCRwas set up in a total volumeof 20mLcontaining 2 mL of cDNA template, 2 mL of 103 PCR buffer (100 mM Tris-HCl, pH8.3, 500mMKCl, 15mMMgCl2, and0.01%gelatin), 2mLof dNTPs(40 mM), 1 mL each of 5 mM forward and reverse primers, 0.1 mL of Hot-StarTaq DNA polymerase (5 U/mL; Qiagen), and 11.9 mL of nuclease-freewater. After purification, PCR product was sequenced using Big DyeTerminator chemistry and an ABI 3730 XL instrument (Life Technologies).The HvCMF7 gene structure was resolved by aligning the sequencedHvCMF7 coding sequence to genomic sequence of barley cv ’Morex’.

Site-Directed Mutagenesis by RNA-Guided Cas9 Endonuclease

The coding region of theHvCMF7 gene of barley cv ‘Golden Promise’wassequenced and used for gRNA/Cas9 target motif selection and gRNAdesign. The search for genomic target motifs was focused to the regionsurrounding the 4-bp deletion carried by the original albostrians mutant,and one target on each side of the deletion was selected through in silicoanalysis using the online prediction tool (https://www.deskgen.com/guidebook/; the ‘KNOCKIN’ panel was chosen for gRNA design; Doenchet al., 2014). A synthetic double-stranded oligonucleotide carrying thetarget-specific part of the gRNA was inserted between the OsU3 (RNApolymerase III) promoter and thedownstreamgRNAscaffold present in themonocot-compatible intermediate vector pSH91 (Budhagatapalli et al.,2016).Next, the fragmentcontaining theexpressioncassettesofgRNAandCas9 was introduced into the binary vector p6i-d35S-TE9 (DNA-Cloning-Service) using theSfiI restriction sites. The vectors constructed for the twotargetmotifswere thenused inAgrobacterium-mediatedcotransformationof barley cv ‘Golden Promise’ for generation of primary mutant plants.Presence/absence of T-DNA in the regenerated plantlets and mutationdetection was achieved by PCR using custom-designed T-DNA–specificand HvCMF7-specific primers, respectively (Supplemental Table 4).

Formaldehyde Agarose Gel Electrophoresis

Electrophoresis was performed under RNase-free conditions: theelectrophoresis chamber and comb were washed with 0.1% (v/v) DEPC-treated water, and the agarose gel, containing 2% (w/v) agarose, 13 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, and 6.29% (v/v)formaldehyde,waspreparedwith0.1%(v/v)DEPC-treatedwater. TheRNAsample (1 to5mg)wasmixedwith formaldehyde loadingdye that contained25mL of formamide (Carl Roth), 5mL of 103MOPSbuffer (200mMMOPS,50 mM sodium acetate, and 10 mM EDTA; Carl Roth), and 10 mL of 37%formaldehyde (Carl Roth) and incubated 5 min at 65°C for denaturation,followed by adding 2 mL of ethidium bromide (10 mg/mL; Carl Roth) to themix and electrophoresis in 13 MOPS buffer at 85 V for 2.5 h. RNA wasvisualized in the gel by excitation under UV light using the BioDocAnalyzegel-analyze system (Biometra).

Transmission Electron Microscopy Analysis

The first leaf of seedlings at 3 d after germination was collected, and threeindependent plants were sampled for each genotype, representing threebiological replicates. Leaf section preparation, fixation, and embeddingfollowed the protocol described previously (Schwarz et al., 2015), withminor modification. Instead of using the Lowicryl HM20 resin, Spurr resin(Sigma-Aldrich)was used for embedding.Ultrathin leaf sections of;70nm

were used for ultrastructural analysis with a transmission electron mi-croscope (Tecnai G2-Sphera 200 kV, FEI).

Subcellular Localization

The GFP gene (Chiu et al., 1996) was introduced into the HindIII/HincIIcloning sites of pUbi-ABM (DNA-Cloning-Service) to form vector pSB179.The cTP domain–encoding region of HvCMF7 or the coding sequence ofHvCMF7 from the wild type (cv ‘Haisa’), albostrians mutant M4205 (cv‘Haisa’), or TILLING mutant 6460-1 (cv ‘Barke’) was fused to sequenceencoding the N terminus of the GFP reporter (Chiu et al., 1996) throughligation into the SpeI/HindIII cloning sites of the vector pSB179, re-spectively. To verify the potential chloroplast localization of HvCMF7, weused the plastid marker pt-rk-CD3-999, which fuses the targeting se-quence (first 79 amino acids) of the small subunit of tobacco ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to the mCherry fluores-cent protein, with the expression cassette driven by a cauliflower mosaicvirus35Spromoterwithdualenhancerelements (d35S).Detailed informationfor the plastidmarker can be foundat https://www.arabidopsis.org/servlets/TairObject?type5stockandid53001623338. The alternative forms ofHvCMF7:GFP plasmids were tested via particle bombardment using PDS-1000/He system with Hepta adaptor particle delivery system (Bio-RadLaboratories). The physical parameters 1100-psi helium pressure and 27-inch Hg vacuum were set to reach efficient bombardment conditions forbarley epidermal cells. Preparation and delivery of DNA-coated gold par-ticles was performed as described previously (Budhagatapalli et al., 2016).Thesamplewas then scanned for thepresenceof fluorescencesignalswithanLSM780confocal laser scanningmicroscope (Carl ZeissMicroImaging).

Agrobacterium-Mediated Barley Transformation

The expression cassette of the intermediate vectorHvCMF7:GFP, used forthebombardment experiment,wascloned into thebinary vector p6i-d35S-TE9 (DNA-Cloning-Service) using the SfiI restriction sites. The derivedvector was designated as pML14. Subsequently, pML14 was used inAgrobacterium-mediated transformation of barley cv ‘Golden Promise’following the protocol described previously (Hensel et al., 2009).

Accession Numbers

The sequence of the HvCMF7 gene is submitted to the European Nu-cleotide Archivewith accession number LT906619. Accession numbers ofBAC assemblies for each sequenced BAC clone are summarized inSupplemental Table 3.

Supplemental Data

Supplemental Figure. Expression profile of HvCMF7 in barley.

Supplemental Table 1. Markers used for genetic mapping.

Supplemental Table 2. Anchoring markers to the physical map ofbarley.

Supplemental Table 3. List of the sequenced MTP BACs.

Supplemental Table 4. Primers used in this study.

Supplemental Table 5. Identified TILLING mutants for HvCMF7.

ACKNOWLEDGMENTS

We gratefully acknowledge M. Ziems, J. Pohl, S. König, and I. Walde (IPK)for their technical support in keeping plant material, performing TILLINGanalyses, and Sanger sequencing; H. Mueller (IPK) for photography;

1442 The Plant Cell

S. Sommerfeld (IPK) for barley transformation; M. Benecke and K. Hoffie(IPK) for their help in electronmicroscopy analysis; K. Lenz andN.Mehlitz(Humboldt University Berlin and IPK) for initial mapping experiments;H. Trautwein (IPK) for help in particle bombardment experiments; andA. Graner, R. Zhou, M. Jost, S. Hiekel, and N. Wendler (IPK) for helpfuldiscussions. The work was supported by a fellowship of the ChinaScholarship Council to M.L.; by Humboldt-Innovation (SK043 to T.B.);by the German Research Foundation (Deutsche Forschungsgemein-schaft) (grant STE 1102/13-1 to N.S. and grant KU 1252/8-1 to J.K.);and core funding of the Leibniz Institute of Plant Genetics and Crop PlantResearch (IPK).

AUTHOR CONTRIBUTIONS

N.S., T.B., and J.K. designed research;M.L., G.H.,M.Me., N.B., T.R., A.H.,andV.K.performedexperiments;M.L.,M.Ma., andS.B. analyzeddata; andM.L., T.B., and N.S. wrote the article.

Received February 26, 2019; accepted April 23, 2019; published April 25,2019.

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Genetic Control of albostrians Variegation 1445

DOI 10.1105/tpc.19.00132; originally published online April 25, 2019; 2019;31;1430-1445Plant Cell

Axel Himmelbach, Sebastian Beier, Viktor Korzun, Jochen Kumlehn, Thomas Börner and Nils SteinMingjiu Li, Goetz Hensel, Martin Mascher, Michael Melzer, Nagaveni Budhagatapalli, Twan Rutten,

BarleyalbostriansGene in Leaf Variegation and Impaired Chloroplast Development Caused by a Truncated CCT Domain

 This information is current as of September 25, 2020

 

Supplemental Data /content/suppl/2019/04/25/tpc.19.00132.DC1.html /content/suppl/2019/07/02/tpc.19.00132.DC2.html

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