genome-wide target mapping shows histone deacetylase ... · genome-wide target mapping shows...

Genome-wide Target Mapping Shows HistoneDeacetylase Complex1 Regulates Cell Proliferation inCucumber Fruit1[OPEN]

Zhen Zhang,a,2 Bowen Wang,b,c,2 Shenhao Wang,a,2 Tao Lin,d,3 Li Yang,e Zunlian Zhao,a Zhonghua Zhang,b

Sanwen Huang,c and Xueyong Yanga,b,3,4

aCollege of Horticulture, Northwest A&F University, Yangling 712100, ChinabKey Laboratory of Biology and Genetic Improvement of Horticultural Crops of Ministry of Agriculture,Sino-Dutch Joint Lab of Horticultural Genomics, Institute of Vegetables and Flowers, Chinese Academy ofAgricultural Sciences, Beijing 100081, ChinacChina Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen518124, ChinadCollege of Horticulture, China Agricultural University, Beijing 100094, ChinaeCollege of Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, China

ORCID IDs: 0000-0002-4864-6180 (Zhen.Z.); 0000-0003-0884-7452 (B.W.); 0000-0003-1422-020X (S.W.); 0000-0002-1034-227X (Zhonghua.Z.);0000-0002-8547-5309 (S.H.); 0000-0001-5894-9217 (X.Y.).

Histone deacetylase (HDAC) proteins participate in diverse and tissue-specific developmental processes by forming variouscorepressor complexes with different regulatory subunits. An important HDAC machinery hub, the Histone DeacetylaseComplex1 (HDC1) protein, participates in multiple protein–protein interactions and regulates organ size in plants. However,the mechanistic basis for this regulation remains unclear. Here, we identified a cucumber (Cucumis sativus) short-fruit mutant(sf2) with a phenotype that includes repressed cell proliferation. SF2 encodes an HDC1 homolog, and its expression is enriched inmeristematic tissues, consistent with a role in regulating cell proliferation through the HDAC complex. A weak sf2 allele impairsHDAC targeting to chromatin, resulting in elevated levels of histone acetylation. Genome-wide mapping revealed that SF2directly targets and promotes histone deacetylation associated with key genes involved in multiple phytohormone pathwaysand cell cycle regulation, by either directly repressing or activating their expression. We further show that SF2 controls fruit cellproliferation through targeting the biosynthesis and metabolism of cytokinin and polyamines. Our findings reveal a complexregulatory network of fruit cell proliferation mediated by HDC1 and elucidate patterns of HDC1-mediated regulation of geneexpression.

Acetylation and deacetylation of Lys residues, me-diated by histone acetyltransferase or histone deace-tylase (HDAC) enzymes, usually leads to relaxedor tight chromatin structures. These are generally as-sociated with transcriptional activation or repres-sion, respectively. A growing number of studies havedemonstrated the critical functions of histone deace-tylation/acetylation in various biological processes,such as genome stability (Shahbazian and Grunstein,2007; Zilio et al., 2014), transcriptional regulation(Rossi et al., 2007; Zhang et al., 2018), development(Ueno et al., 2007; Cigliano et al., 2013; Liu et al., 2014),and cell division control (Kouzarides, 1999; Jamaladdinet al., 2014). In animals, HDAC1 and HDAC2 have es-sential roles in cell proliferation and regulate stemcell self-renewal (Jamaladdin et al., 2014). Similarly, inplants, it has been shown that histone deacetylationactivity controls root meristem cell division (Ikeuchiet al., 2015). Plant cell division is a continuous processin apical meristematic cells or quasi-meristematic cells,which generate new cells throughout the life of theplant (Girin et al., 2009). However, as HDAC enzymes

are generally ubiquitously expressed, the regulationof histone deacetylation activity during cell divisionis currently not well understood. To carry out theirintended functions, the correct assembly of HDACproteins into various types of functional complexes, con-taining different regulatory subunits, is important fortheir diverse, and often cell-specific, roles (Utley et al.,1998; Gonzalez et al., 2007).Recent studies in Arabidopsis (Arabidopsis thaliana)

identified HISTONE DEACETYLASE COMPLEX1(HDC1), which contains a yeast regulator of transcrip-tion3 domain, as an important subunit of the plantSWI-INDEPENDENT3 (SIN3)–HDAC machinery. InArabidopsis, HDC1 functions as a hub to enable mul-tiple protein interactions in HDAC complexes. Ge-netic analyses showed that HDC1 regulates abscisicacid (ABA) sensitivity and promotes increases in or-gan size (Perrella et al., 2013). Because overexpressionof HDACs has been reported as being either ineffec-tive, or as causing pleiotropic morphological abnor-malities (Tian and Chen, 2001; Tian et al., 2005; Longet al., 2006), the regulation of organ size is thought to

Plant Physiology�, January 2020, Vol. 182, pp. 167–184, www.plantphysiol.org � 2020 American Society of Plant Biologists. All Rights Reserved. 167

https://plantphysiol.orgDownloaded on January 31, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

http://orcid.org/0000-0002-4864-6180

http://orcid.org/0000-0002-4864-6180

http://orcid.org/0000-0003-0884-7452

http://orcid.org/0000-0003-0884-7452

http://orcid.org/0000-0003-1422-020X

http://orcid.org/0000-0003-1422-020X

http://orcid.org/0000-0002-1034-227X

http://orcid.org/0000-0002-1034-227X

http://orcid.org/0000-0002-8547-5309

http://orcid.org/0000-0002-8547-5309

http://orcid.org/0000-0001-5894-9217

http://orcid.org/0000-0001-5894-9217

http://orcid.org/0000-0002-4864-6180

http://orcid.org/0000-0003-0884-7452

http://orcid.org/0000-0003-1422-020X

http://orcid.org/0000-0002-1034-227X

http://orcid.org/0000-0002-8547-5309

http://orcid.org/0000-0001-5894-9217

http://crossmark.crossref.org/dialog/?doi=10.1104/pp.19.00532&domain=pdf&date_stamp=2019-12-31

https://plantphysiol.org

be regulated by HDC1 through site- or tissue-specificutilization of certain HDAC complexes. One studyshowed that HDC1 mRNA is ubiquitously expressedin all vegetative tissues in Arabidopsis (Perrella et al.,2013); however, it has yet to be determined whetherthe HDC1 protein accumulates in meristematic cells,thus enabling the function of the HDAC complexin cell division. Moreover, the genome-wide targetsof HDC1 are still unknown, and so the regulatorymechanism by which HDC1 controls plant organ sizeis unclear.

The identities, interactions, and control of the manyfactors that dictate plant organ size and shape, whichin turn are determined by cell number and size, areimportant ongoing questions. Fruit size and shape areimportant agronomic traits that are associated withcrop yield. Classic fruit morphogenesis is divided intofour physiological phases during early development:ovary growth, fruit set, rapid cell proliferation, andsubsequent cell expansion (Ando and Grumet, 2010;Yang et al., 2013; Grumet and Colle, 2016). It is thoughtthat the cell proliferation, and therefore the final sizeand shape of the fruit, is dependent on initial meriste-matic and quasi-meristematic characteristics (Girinet al., 2009). As morphologically complex fruit cantake days or weeks to develop, the establishmentof the quasi-meristem in the medial part of the fruittissue allows for prolonged cell division after fruitset. Therefore, fruit organ growth is fine-tuned bymaintenance of these medial tissues and their “quasi-meristematic” fate. However, although many quanti-tative trait loci related to fruit size have been identified

(Qi et al., 2013; Wei et al., 2014; Bo et al., 2015; Wenget al., 2015; Pan et al., 2017), the genetic and molecularmechanisms that regulate rapid cell proliferation dur-ing fruit morphogenesis remain largely unclear.

Fleshy cucurbit fruits initiate from female floralmeristems, and are known for their extreme diversityin shape (oblate to elongate) and size (Grumet andColle, 2016; Colle et al., 2017). Here, we identified re-cessive allelic variation in a cucumber (Cucumis sativus)HDC1 homolog, Short Fruit2 (SF2). This weak mutationsignificantly represses fruit elongation by reducing cellproliferation by 70%. In contrast to a previous reportconcluding that it is ubiquitously expressed in all veg-etative tissues, we found that SF2 protein specificallyaccumulates in meristematic tissues undergoing cellproliferation. We further show that the SF2 protein isexpressed during early fruit development and accu-mulates in the early placenta, replum, and ovules,which are thought to maintain quasi-meristematic ac-tivity during early fruit development (Girin et al., 2009).Genome-wide analysis showed that SF2 directly targetsand promotes histone deacetylation of genes involvedin multiple phytohormone pathways and cell cycleregulation, and represses or activates their expressionduring cell proliferation. We reveal that HDC1 controlsfruit cell proliferation through direct targeting of cyto-kinin (CK) and polyamine (PA) biosynthesis and me-tabolism. These findings enhance our understandingof the role of histone deacetylation in cell proliferationand fruit morphogenesis.

RESULTS

A Short-Fruit Phenotype Is Determined by a SingleNucleotide Change in Cucumber HDC1

We identified a cucumber ethyl methanesulfonatemutant bearing short fruit (named “sf2”) that was oth-erwise phenotypically similar to the wild type (Fig. 1,A–C). Preliminary genetic analysis showed that all F1plants from a sf2 3 wild type cross exhibited a wild-type phenotype, and the selfed F2 progeny segregatedat an;3:1 ratio (249:78, wild type to mutant; x25 0.23),indicating that the short-fruit phenotype is determinedby a single recessive gene (Fig. 1D; Supplemental DataSet S1).

Further characterization of fruit morphogenesisduring early fruit development indicated that the wild-type fruit exhibited more rapid growth than sf2 fruitafter anthesis, while there was no significant differencein fruit diameter (Fig. 1, E and F; Supplemental Fig. S1).The cell proliferation rate and duration of this processwere both notably reduced in sf2 fruit compared withwild type in the longitudinal direction, resulting in;70% fewer cells in the sf2 mutant at 8 days after an-thesis (DAA; Fig. 1G). Despite a potential compensa-tory mechanism resulting in an increase in cell size tomake up for the reduction in cell numbers, sf2 fruitlength was reduced by 50% (Fig. 1, H–J), demonstrating

1This work was supported by grants from the National NaturalScience Foundation of China (31530066 to S.H. and 31572117 to X.Y.),the Fundamental Research Funds for the Central Universities(2452019048 to S.W.), the National Natural Science Foundation ofChina (313220419 to Z.H.Z.), the National Key R&D Program ofChina (2016YFD0101007 and 2016YFD0100500), and the Central Pub-lic-interest Scientific Institution Basal Research Fund (No.Y2017PT52). Additional support was provided by the Chinese Acad-emy of Agricul tural Sc ience (ASTIP-CAAS and CAAS-XTCX2016001), the Leading Talents of Guangdong Province Program(00201515 to S.H.) and the Shenzhen Municipal (The Peacock PlanKQTD2016113010482651) and the Dapeng district government.

2These authors contributed equally to this article.3Senior authors.4Author for contact: [email protected] author responsible for distribution of materials integral to the

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

X.Y., S.H., and Zhen.Z. designed the research; Zhen.Z., S.W., andX.Y. performed the mutant screen, genetic studies, and phenotypeobservations; Zhen.Z., X.Y., and B.W. made major contributions tobiochemical analyses and Co-IP assays; B.W., Zhen.Z., and X.Y. per-formed the ChIP assay, the LCI assay, and the CKX enzyme activityassay; T.L., X.Y., and Zhonghua.Z. led bioinformatic analyses; S.W.,X.Y., L.Y., and Z.L.Z. led genetic transformation of plants; X.Y., S.H.,S.W., and Zhen.Z. interpreted the data and wrote the article.

[OPEN]Articles can be viewed without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.19.00532

168 Plant Physiol. Vol. 182, 2020

Zhang et al.


http://www.plantphysiol.org/cgi/content/full/pp.19.00532/DC1



http://www.plantphysiol.org/cgi/doi/10.1104/pp.19.00532


that the sf2 phenotype primarily involves reduced cellproliferation, which results in shorter fruit lengthcompared with wild type.To clone the SF2 gene, we used a combination of

the “MutMap” strategy (Abe et al., 2012) and tradi-tional linkage analysis in an F2 population. We identi-fied only one single-nucleotide polymorphism (SNP;2G15231244) that cosegregated with the sf2 locus asthe causative SNP (Fig. 1, K and L; Supplemental DataSet S2). The nonsynonymous SNP 2G15231244 causeda G-to-A substitution within Csa2G337260, resulting inan amino acid change fromGly (G) to Glu (E) at residue515 (G515E). Csa2G337260 encodes an HDC1 homolog(Fig. 1, M and N; Perrella et al., 2013), a subunit of theHDAC complex (Perrella et al., 2016).To demonstrate the function of Csa2G337260, we

first knocked out SF2 expression by clustered regu-larly interspaced shot palindromic repeat/CRISPRassociated protein 9 (CRISPR/Cas9; Fig. 2A) to pro-duce CR-sf2 mutants containing small deletionsthat produce a frameshift in the SF2 coding sequenceresulting in knock-out of SF2 protein. We observed aserious growth inhibition of the entire CR-sf2 shootcompared with the control plants transformed with

an empty vector. Seedlings did not regenerate fromthese shoots (Fig. 2, B–E). Consistent with the obser-vation that the hdc1 mutant in Arabidopsis showed a50% reduction in fresh weight compared with wildtype (Perrella et al., 2013), our study suggested ageneral function of plant HDC1 in controlling meri-stematic cell proliferation.To confirm the identity of Csa2G337260 as SF2, a

DNA sequence containing a ;1-kb region of the nativepromoter plus the full-length coding sequence of wild-type Csa2G337260was transformed into the sf2mutant.Both of the homozygous complemented plants dis-played increased cell numbers and fruit length (Fig. 2,F–H; Supplemental Fig. S2). We therefore concludedthat Csa2G337260 is responsible for the observed short-fruit phenotype of sf2.

The SF2 Protein Is Specifically Expressed inMeristematic Tissues

Although a previous study in Arabidopsis reportedthat HDC1 is ubiquitously expressed in vegetative tis-sues (Perrella et al., 2013), we hypothesized that the

Figure 1. Characterization and cloning of the SF2 gene. A to C, Phenotypic analysis of wild-type (WT) cucumber line 406 and sf2mutant plants. A, Vegetative growth and development. Scale bar5 5 cm. B, Leaf and flower development. Scale bar5 5 cm. Imageswere digitally abstracted and made into a composite for comparison. C, Seed development. Scale bar5 1 cm. D, Fruit of WT (406background), sf2, and their F1 plants at 16 DAA. Scale bar5 5 cm. E, Fruit length of WTand sf2 during fruit development. Bars5means6 SE of three replicates. F, Fruit diameter ofWTand sf2 during fruit development. Bars5means6 SE of three replicates.G, Cellproliferation during fruit development in WT and sf2. Bars 5 means 6 SE of three replicates. H, Average cell size during fruit de-velopment inWTand sf2. Bars5means6 SE of three replicates. I,WTmesocarp cells at 16DAA. Scale bar5 50mm. J, sf2mesocarpcells at 16 DAA. Scale bar5 50 mm. K, SNP-index distribution of the mutant pool. The arrow and pink rectangle indicate the regionon chromosome 2with a SNP index. 0.5. L, Linkage analysis of the F2 population using dCAPSmarkers. The red arrow indicates theonly SNP cosegregating with the short-fruit phenotype. S, short fruit; L, long fruit. M, Csa2G337260 gene structure. N, Alignment ofCsa2G337260 homologs from different species, highlighting G515E mutation in sf2 in red.

Plant Physiol. Vol. 182, 2020 169

Epigenetic Regulation of Cell Division






HDC1 protein may be specifically enriched in meriste-matic tissues. To test this, we generated an SF2 poly-clonal antibody (Fig. 3, A–C), which we used to detectHDC1 accumulation in various tissues. We found thatin contrast to the ubiquitous expression of SF2 mRNA,the SF2 protein was indeed specifically detected inmeristematic tissues in which rapid cell proliferationwas occurring, including: root tip at 1 d after germi-nation (1-d root tip); stem tip; leaves measuring 5 mmin width (5-mm leaf); female flowers (23 DAA ofovary); and placenta of fruit on the day of anthesis (0DAA of fruit; Fig. 3D). This result was consistent with arole for SF2 in regulating cell proliferation.

To investigate a potential correlation between SF2expression and cell proliferation, we investigated the ex-pression pattern of SF2 mRNA and protein during fruitdevelopment. SF2 mRNA exhibited highly similar ex-pression levels in wild type and sf2mutants in the stagesinvestigated (Fig. 3E), while the SF2 protein specificallyaccumulated in the fruit during stages of rapid cell divi-sion (28 DAA to 3 DAA; Fig. 3F). These results alsosuggested that posttranscriptional regulation of SF2 re-sults in spatially restricted SF2 accumulation in the tissuesundergoing exponential cell division. We performedimmunolabeling of fruit sections using the SF2 antibodies,

which revealed that the SF2 protein primarily accumu-lates in the early placenta, replum, andovules (Fig. 3G), allof which maintain quasi-meristematic activity and allowfruit cell proliferation.

SF2 Targets Active Genes and PromotesHistone Deacetylation

In Arabidopsis, HDC1 interacts with several com-ponents of the HDAC complex, such as HDA6,HDA19, SIN3-associated protein18 (SAP18), MULTI-COPY SUPPRESSOR OF IRA (MSI), and SIN3-LIKE(Perrella et al., 2016). We confirmed these interactionsfor SF2 in cucumber (Supplemental Fig. S3, A and B;Supplemental Data Sets S3–S6), and found SF2 coim-munoprecipitated with HDA19A, HDA19B, SIN3-LIKE1, SIN3-LIKE3, SAP18, and MSI1 homologs bycoimmunoprecipitation (Co-IP) and liquid chroma-tography with tandem mass spectrometry (LC-MS/MS). We further confirmed that SF2G515E shows animpaired capacity for interaction with SIN3-LIKE1 andSIN3-LIKE3 by luciferase complementation imaging(LCI) assays andCo-IP assay (Supplemental Figs. S3C, S4,and S5).

Figure 2. Genetic verification of SF2function in vivo. A, Specific target on thefirst exon of the SF2 gene was selected.The deletion alleles (CR-sf2-1 and CR-sf2-2) were detected by PCR and se-quencing. The sgRNA target sequence ishighlighted in red and the protospacer-adjacent motif (PAM) site is underlined.B, The regenerated shoots of CR-sf2-1 and control plants expressing a vectorcontrol. Scale bar 5 5 cm. C, Theregenerated positive shoots displayingGFP fluorescence. Scale bar5 2 mm. Dand E, The regenerated positive shoots ofthe vector control (D) and CR-sf2 (E). Forbright field images, scale bar5 5 cm; forGFP fluorescence, scale bar 5 2 mm. Fand G, The fruit phenotype of the com-plemented plants. Scale bar 5 5 cm.Bars 5 means 6 SE of three replicates.H, Cell numbers of fruits in two com-plemented COM-1 and COM-2 plants.Scale bar 5 50 mm. WT, wild type.


Zhang et al.









Binding of the SIN3-HDAC complex to chromatin isaccomplished by flexible interactionswithDNA-bindingproteins, primarily through SIN3 repressors (Grzendaet al., 2009). Therefore, we reasoned that the capacity ofHDAC complex targeting to genomic regions might bedecreased in the sf2mutant, and that the relative histoneacetylation levels might be increased. To test this, chro-matin immunoprecipitation (ChIP)-sequencing (Seq)assays were performed using the SF2 antibody (Fig. 4A).By comparing the genome-wide binding profiles of SF2inwild-type and sf2mutant fruits, we found that the SF2-HDAC complex was primarily detected at the tran-scriptional start site (TSS) and in gene body regions(Fig. 4, A and B), which is similar to the pattern of humanHDAC6 (Wang et al., 2009). Indeed, the SF2-HDACcomplex showed strongly decreased binding levels in thesf2 mutant compared to the wild type (Fig. 4A). Wefurther analyzed the genomic distribution of over-lapping SF2 binding sites in two replicates (Fig. 4B;Supplemental Data Set S7), and identified 3,356 genes asputative SF2 target genes (Supplemental Fig. S6, A–C;Supplemental Data Set S8).

To investigate the correlation between SF2 bindingand gene expression, we divided all cucumber genesinto four groups based on their expression levels (high,medium, low, and silent expression), and correlatedthis grouping with SF2 binding levels (Fig. 4C). Thisrevealed that SF2 is preferentially enriched in activegenes compared to silent genes (Fig. 4C). As HDACcomplexes are generally considered to be transcrip-tional repressors, SF2 enrichment in highly expressedgenes might be considered counterintuitive. Neverthe-less, studies of HDACs in both mammals and plantshave yielded similar results, suggesting a conservedaction of the HDAC complex on active genes (Wanget al., 2009; Yang et al., 2016).We next searched for putative DNA-binding motifs in

SF2 binding peaks using the software “MEME 4.0”(Bailey et al., 2006) and identified three significantlyenriched consensus motifs (Supplemental Fig. S7, A–C).Comparison of these motifs against a database of knownmotifs, using TOMTOM (http://meme-suite.org/tools/tomtom), suggested that motif 1 andmotif 2 are similar tothe binding motif of the GATA1 transcription factor

Figure 3. Expression pattern of the SF2 protein. A, Schematic presentation of the SF2 protein, showing the 515th amino acidresidue changed from aGly residue (G) to a Glu residue (E). The 17-residue PMSKIPRTESRDGDRRS peptide at the SF2N-terminalwas selected as an antigen for making a polyclonal antibody. B, SDS-PAGE and immunoblot analysis of wild-type (WT) fruits at 0,5, and 8 DAA using the anti-SF2 polyclonal antibody. Left: The antibody recognized a specific band in cucumber fruits. Right:CBB, Coomassie brilliant blue staining as loading control. C, The pCAMBIA 1300-c-Myc-SF2 and the corresponding empty vectorwere transiently expressed in N. benthamiana leaves. Total protein was extracted for immunoblotting using the anti-SF2 andantic-Myc antibodies. D, Expression pattern of SF2mRNA and its encoded protein in different tissues. RT-PCR analysis shows theexpression of SF2 in root, stem, cotyledon, leaf, male flower, female flower, pericarp (Pe), and placenta (Pl) of 0-DAA fruit and 16-DAA fruit and tendrils. UBQ is used as a reference gene. SF2 protein was detected by immunoblotting in various plant tissues. E,SF2 expression in WTand sf2 fruits at 28, 25, 23, 0, 3, 5, 8, and 16 DAA. Bars 5 means 6 SE of three replicates. F, Expressionpattern of the SF2 protein during fruit development. Actin was used as an internal control. G, Immunolabeling assays of wild-type(WT) fruits using the SF2 antibody. Scale bars 5 5 cm (F) and 500 mm (G).










http://meme-suite.org/tools/tomtom

http://meme-suite.org/tools/tomtom


(T/CCDT/CCNT/CCNT/CCN, P 5 4.14e-06;CANCANCAN, P 5 9.71e-03; Supplemental Fig. S7, Aand B). In mouse (Mus musculus), the HDAC complexphysically interacts with GATA to modulate the ex-pression of cell cycle genes during embryonic develop-ment (Trivedi et al., 2010). The third motif was similar tothe binding site of high-mobility–group box protein(CC/TDCC/TDCC/TD, P 5 4.98e-07; SupplementalFig. S7C), which has been implicated in interacting withmitotic andmeiotic chromosomes (Pedersen et al., 2011).

To investigate the relationship between SF2 bindingand histone acetylation, a global analysis of H3K9 andH3K14 acetylation status in wild-type and sf2 mutantfruits was conducted (Supplemental Fig. S8; SupplementalData Set S9). The level of SF2 binding increased with in-creasingH3K9ac andH3K14ac (Fig. 5, A andB), indicatingthat SF2 preferentially targets genes with high histoneacetylation levels. To better understand how the impairedbinding of the HDAC complex in the sf2 mutant affectshistone acetylation levels, we performed a genome-widecomparison of H3K9ac andH3K14ac levels in wild typeand sf2 and observed that sf2 showed increasedH3K9ac and H3K14ac levels (Supplemental Fig. S9;Supplemental Data Sets S10 and S11). We furthercompared the normalized H3K9ac and H3K14ac levelsaround the SF2 binding sites in wild type and sf2 mu-tant (Fig. 5, C and D). The results showed that com-pared with wild type, H3K9ac, and H3K14ac levelsincreased in most regions of promoters and gene bodiesin sf2mutant, demonstrating that SF2 promotes histonedeacetylation at its binding sites. Accordingly, immu-noblotting demonstrated that the levels of histone H3acetylation increased in 0 DAA of sf2 fruit (Fig. 5E),especially at Lys residues K9 and K14 (H3K9ac andH3K14ac), demonstrating that SF2 promotes histonedeacetylation.

Identification of The Genetic Network Activated orRepressed by SF2

To obtain a detailed understanding of SF2 functionin target gene regulation, we used RNA-Seq analysis

to identify genes that are differentially expressedin 0-DAA fruits between wild-type and sf2 plants(Supplemental Data Sets S12 and S13). Gene ontology(GO) analysis revealed that the 1,494 upregulated genesin the sf2 mutant are primarily involved in photosyn-thesis and phytohormone responses (Supplemental Fig.S10A); and that the 1,272 downregulated genes in sf2are enriched in the “cell cycle,” “cytokinesis,” and“DNA replication” terms (Supplemental Fig. S10B),which is consistent with the reduced cell number phe-notype of sf2. The RNA-seq data were validated byreverse-transcription quantitative PCR (RT-qPCR)analysis. All the 10 selected genes involved in phyto-hormone biosynthetic and signaling pathways and alsocell cycle regulation showed expression patterns cor-responding with the RNA-seq data (Supplemental Fig.S10, C and D).

Because histone deacetylation is generally associatedwith repressed gene expression, to map the gene net-work directly repressed by SF2, the 3,356 target geneswith potential SF2 binding sites were compared withthe 1,494 genes upregulated in sf2. A total of 321 genesshowed an overlap between the two data sets (Fig. 6A;Supplemental Data Set S14). To determine whether SF2promotes deacetylation of these 321 core repressedgenes, the genes with hyper-H3K9ac and hyper-H3K14ac (Supplemental Data Sets S10 and S11) weretested for overlap with the core repressed genes. Thisanalysis revealed that 108 and 140 of the core SF2-repressed genes were hyperacetylated at H3K9 andH3K14, respectively, in the sf2 mutant, in either thegene body or in the promoter (Fig. 6, B and C;Supplemental Data Sets S14–S16). Furthermore, 52genes had both hyper-H3K9ac and hyper-H3K14acsites (Fig. 6D; Supplemental Data Set S17), while 125out of the 321 genes did not show significantly in-creased H3K9ac/K14ac in the sf2 mutant (Fig. 6D),suggesting a possible modification of other Lys resi-dues (e.g. K18, K23, K27, and K56; Fig. 5E).

Among the 321 core repressed genes, we found astrong enrichment of genes involved in hormone bio-synthesis and signal transduction (Fig. 6E; SupplementalFig. S11; Supplemental Data Set S18), indicating a role for

Figure 4. SF2 binds to active genes. A, Metaplot of the SF2 peak distribution on all genes in 0-DAAwild-type and sf2 fruits. TTS,Transcription Termination Sites;21 k and11 k represent 1 kb upstream of TSS and 1 kb downstream of TTS, respectively. The yaxis represents the normalized read density relative to input DNA. B, Proportion of SF2 target peaks in different parts of thegenome. C, Profiles of SF2 binding levels on genes with high, medium, low, and silent expression.


Zhang et al.


























SF2 in coordinating the expression of genes involved inphytohormone pathways (Fig. 6E). Among these genes,several are known to encode negative regulators ofauxin, gibberellic acid (GA), and CK responses or bio-synthesis (Fig. 6E; Supplemental Data Set S18). For ex-ample, PINOID (Csa1G537400) and PHYTOCHROMERAPIDLY REGULATED (Csa6G423410) mediate auxintransport (Friml et al., 2004) and signaling repression(Bou-Torrent et al., 2008), respectively. GIBBERELLININSENSITIVE (Csa1G408720) encodes aDELLAproteinthat represses the GA response (Murase et al., 2008) andcytokinin oxidase/dehydrogenase (CKX; Csa4G647490)encodes a CK oxidase/dehydrogenase involved in CKdegradation (Werner et al., 2001). The core repressedgenes also included some positive regulators of ABA,jasmonic acid (JA), and ethylene responses (Fig. 6E;Supplemental Data Set S18), including an ABA1 ho-molog (Csa2G277050) encoding a zeaxanthin epoxidaseinvolved in ABA biosynthesis (Xiong et al., 2002),a PYRABACTIN RESISTANCE-LIKE4 homolog(Csa3G730890) encoding an ABA sensor (Park et al.,2009; Perrella et al., 2013), a myelocytomatosis oncogenehomolog 2 (MYC2) gene (Csa3G902270) that positivelyregulates the JA response, and several ETHYLENERESPONSE FACTOR transcription factor genes thatparticipate in ethylene responses. The repression ofnegative regulators from the auxin, GA, and CK path-ways, and positive regulators from the ABA, JA, andethylene pathways, suggests activation of auxin, GA,and CK pathways and repression of ABA, JA, andethylene pathways by SF2.PAs, which are involved in complex crosstalk with

phytohormones, also play roles in regulating cell divi-sion (Kaur-Sawhney et al., 2003). Our analysis showedthat SF2 targets and represses the expression of several

S-adenosyl-L-Met decarboxylase (SAMDC) genes(Csa2G036680, Csa3G271350, and Csa3G271360) thatencode key enzymes in the biosynthesis of PAs (Anwaret al., 2015). ChIP-qPCR and RT-qPCR assays of 15 se-lected genes involved in phytohormone biosynthesisand signaling pathways further verified these results(Fig. 6, F–I; Supplemental Fig. S12; Supplemental DataSet S18).Although histone deacetylation generally represses

gene expression, research in mice found that a specificsubset of mouse genes could be deregulated in the ab-sence of HDAC1, suggesting a novel function forHDAC1 as a transcriptional coactivator (Zupkovitzet al., 2006). To assess the possibility that SF2 pro-motes histone deacetylation and activates a target genenetwork, the target genes with SF2 binding sites werecompared with the 1,272 genes downregulated in sf2. Atotal of 237 genes showed overlap between the two datasets (Fig. 7A; Supplemental Data Set S19). We furtherfound that 100 and 86 genes were hyperacetylated atH3K9 and H3K14, respectively, in sf2 (Fig. 7, B and C;Supplemental Data Sets S19–S21), of which 43 geneshad both hyper-H3K9ac and hyper-H3K14ac sites(Fig. 7D; Supplemental Data Set S22).GO analysis revealed that the core activated genes

were primarily involved in DNA replication, chromatinassembly, and the cell cycle (Fig. 7E), including TU-BULIN6 (Csa1G629200; Pastuglia et al., 2006), the cyclingene CYCA1;1 (Csa6G382370; de Jager et al., 2005), andCell Division Cycle 20.1 (Csa5G141120; SupplementalData Set S19; Niu et al., 2015). We also found a micro-tubule motor kinesin gene, KF3 (Csa4G002000), whichwas previously implicated in rapid cell division byYang et al. (2013). In addition, a small subset of genes inthe core activated gene network were also involved in

Figure 5. SF2 promotes histone deacetylation. A and B, The relationship between SF2 binding levels and H3K9ac levels (A) orH3K14ac levels (B) among the SF2 target genes. C andD,Metaplots of H3K9ac (C) andH3K14ac (D) around the SF2 binding sitesin 0-DAAwild-type and sf2 fruits. TTS, Transcription Termination Sites;21 k and 11 k represent 1 kb upstream of TSS and 1 kbdownstream of TTS, respectively. The y axis represents the normalized read density of H3K9ac or H3K14ac relative to input DNA.E, The levels of histone H3 acetylation between wild type (WT) and sf2 fruit detected by immunoblotting with antibodies specificfor different acetylated sites.
















Figure 6. Mapping the core genes repressed by SF2. A, Venn diagram showing the overlap between SF2 target genes (left) andgenes upregulated in sf2 (right). The overlapping gene set was designated as core SF2-repressed genes. B and C, Venn diagramsshowing the overlap between genes bound by theHDAC complex, genes upregulated in the sf2mutant, and hyper-H3K9ac genes(B) or hyper-H3K14ac genes (C) from the sf2 mutant. D, Venn diagram showing the status of hyper-H3K9ac or hyper-H3K14acgenes in the sf2mutant among the 321 core SF2-repressed genes. E, Diagram showing the auxin, CK, PA, ethylene, JA, ABA, andGA synthesis and signaling pathways. The components in red font are encoded by the core SF2-repressed genes. Arrows and bar-ended lines represent activation and inhibition, respectively, of which the synthesis pathways are in green and signaling pathwaysare in black; rsp, response. F to H, The SF2 binding level (F), H3K9ac level (G), and H3K14ac level (H) in six core SF2-repressedgenes were determined by ChIP-qPCR. ChIP was performed in 0-DAA wild-type and sf2 fruits with a SF2 polyclonal antibody,anti-H3K9ac, and anti-H3K14ac. I, Relative mRNA expression levels of six core SF2-repressed genes in 0-DAA wild-type (WT)and sf2 fruits detected by RT-qPCR.UBQwas used as internal control. Bars5means6 SE of three replicates. **P, 0.05; ***P,0.01 (t test, one tail).


Zhang et al.



Figure 7. Mapping the core genes activated by SF2. A, Venn diagram showing the overlap of SF2 target genes (left) and genesdownregulated in sf2 (right). The overlapping gene set was designated as core SF2-activated genes. B and C, Venn diagramsshowing the overlap between genes bound by the HDAC complex, genes downregulated in the sf2 mutant, and hyper-H3K9acgenes (B) or hyper-H3K14ac genes (C) from the sf2 mutant. D, Venn diagram showing the status of hyper-H3K9ac or hyper-H3K14ac genes in the sf2 mutant among the 237 core SF2-activated genes. E, GO enrichment analysis of the 237 coreSF2-activated genes. P value, 0.05. F to H, The SF2 binding level (F), H3K9ac level (G), and H3K14ac level (H) of six of the coreSF2-activated genes were determined by ChIP-qPCR. ChIP was performed in 0-DAA wild-type (WT) and sf2 fruits with a SF2polyclonal antibody, anti-H3K9ac, and anti-H3K14ac. I, Relative mRNA expression levels of six core SF2-activated genes in 0-DAAWTand sf2 fruits detected by RT-qPCR.UBQwas used as internal control. Bars5means6 SE of three replicates. **P, 0.05;***P , 0.01 (t test, one tail).





phytohormone biosynthesis or signaling pathways.Some are known to encode positive regulators of GAand CK, including GIBBERELLIN INSENSITIVEDWARF1 (Csa7G391240), which encodes a GA receptor(Griffiths et al., 2006), and LONELY GUY5 (LOG5;Csa6G127300), which is involved in CK biosynthesis(Tokunaga et al., 2012). Some are negative regulators ofABA signaling, such as an ABA-INSENSITIVE1 ho-molog (Csa1G574880; Yoshida et al., 2006). ChIP-qPCRand RT-qPCR assays of 15 selected genes involved incell cycle regulation and phytohormone pathwayswereused to verify these results (Fig. 7, F–I; SupplementalFig. S13). Consistent with the results for the core re-pressed genes, the activation of positive regulators in-volved in the GA and CK pathways, and negativeregulators involved in the ABA pathway, suggested theactivation of GA and CK pathways and repression ofthe ABA pathway by SF2.

To distinguish whether the core activated genes re-quire the HDAC complex directly or indirectly for theiractivation, we analyzed several candidate genes fortheir responsiveness to Trichostatin A (TSA), a deace-tylase inhibitor. We can conclude that genes requiringHDAC complexes for their direct activation should benegatively regulated by the deacetylase inhibitor inwild type. As shown in Supplemental Fig. S14, the fourcore activated genes bound by SF2 were verified byChIP-qPCR. We showed that after treatment with TSAfor 10 h, in both wild type and sf2 mutant, on most ofthese genes, the H3K9ac and H3K14ac levels were sig-nificantly increased, and their mRNA expression levelsdecreased. A recent study in mammalian cells showedthat HDACs are required for limiting acetylation ingene bodies, and this function facilitates efficient tran-scriptional elongation (Greer et al., 2015). Our resultsindicated that in plants, the HDC1-HDAC complexprobably can also activate the core genes activated byhistone acetylation/deacetylation.

Because CK and GA signaling cascades mediate cellproliferation, while ABA generally has the oppositeeffect, these results suggest a function of SF2 in regu-lating cell proliferation by direct activation of the CKand GA signaling pathways and repression of ABAsignaling.

Expression of SF2 Core Targets Occurs in Regions ofCell Proliferation

To further verify whether the SF2 core target genesare indeed involved in cell proliferation, we relatedtheir expression patterns in our transcriptome datawithcell proliferation during fruit development.

Given that SF2 promotes cell division, its core re-pressed genes should be implicated in repression of cellproliferation, and thus, negatively correlate with cellproliferation during fruit development. Our resultsshowed that the 321 core repressed genes could begrouped into three clusters, based on their expressionpatterns. The genes in both cluster 1 (126 genes; 39%)

and cluster 2 (110 genes; 34%) showed reduced ex-pression levels during exponential cell proliferation(0–3 DAA), whereas only 77 genes belonged to cluster 3(24%), showing higher expression levels during cellproliferation (0–3 DAA; Fig. 8A; Supplemental Data SetS23). This indicated that the expression patterns of mostof the core repressed genes were negatively related tocell proliferation. RT-qPCR analysis of ABA1, GIB-BERELLIN INSENSITIVE, CKX7, SAMDC1, ETHYL-ENE RESPONSE FACTOR, and SAMDC2 confirmedthat their expression was significantly repressed by SF2during fruit development (Fig. 8B).

Additionally, the 237 core activated genes weregrouped into three clusters based on their expressionpatterns. The genes in cluster 1 (114 genes; 49%)showed elevated expression levels during exponentialcell proliferation (0–3 DAA), and most of the genes incluster 3 (40 genes; 17%) showed gradually increasedexpression levels after 0 DAA (Fig. 8C). The expressionpatterns of these core activated genes were positivelyrelated to cell proliferation. However, the genes incluster 2 (81 genes; 35%) showed higher expressionlevels at 23 DAA, suggesting specific roles before an-thesis (Fig. 8C; Supplemental Data Set S24). RT-qPCRanalysis of MAP65-6, CYCA 1;1, ABA-INSENSITIVE1,GIBBERELLIN INSENSITIVE DWARF1, LOG5, andTUBULIN6 confirmed that their expression was acti-vated by SF2 (Fig. 8D).

Taken together, these results support the notion thatthe direct target gene networks of SF2 are involved inregulating cell proliferation during fruit development.

SF2 Regulates Cell Proliferation through its Effect on CKand PA Homeostasis

During early fruit development, SF2 activates LOG5but represses CKX7 (Fig. 8, B and D), suggesting a rolein promoting CK synthesis. SF2 also activates the ex-pression of SAMDC genes (Fig. 8B). SAMDC is a keyenzyme in PA biosynthesis of compounds such asspermidine and spermine, which may interact with CKand play essential roles in diverse growth and devel-opmental processes (Kaur-Sawhney et al., 2003). PAsand CKs are essential for cell division (Kaur-Sawhneyet al., 2003; Anwar et al., 2015). This suggests that SF2facilitates cell proliferation through modulation of CKand PA homeostasis by targeting metabolic and bio-synthetic genes. To test this hypothesis, we measuredCKX enzyme activity and CK content in 0-DAA fruitsof wild type and sf2, and showed that the enzymeactivity was significantly elevated in the mutant(Fig. 9A), while the content of isopentenyladenine (iP)and dihydrozeatin decreased (Fig. 9B). Exogenoustreatment of the 23 DAA of female flowers with thi-diazuron (N-phenyl-N9-1,2,3-thiadiazol-5-yl urea, TDZ),an inhibitor of CKX (Hare and Van Staden, 1994), par-tially complemented the short-fruit phenotype (Fig. 9, CandD), and the treatment had amuch stronger effect onthe sf2mutant than onwild-type fruit. This is consistent


Zhang et al.









with a role for SF2 in facilitating cell proliferation throughmodulation of CK contents via targeting of its metabolic(CKX7) and biosynthetic genes (LOG5; Fig. 9E).We next measured PA content and found that the

spermidine levels were significantly higher in sf2 thanin wild type (Fig. 9F). We then sprayed the 23 DAA offemaleflowerswithmethyl-glyoxyl-bis guanylhydrazone(MGBG), a competitive inhibitor of SAMDC, and foundthat the treatment partially complemented the short-fruitphenotype (Fig. 9, G and H) with no significant effect oncell size (Fig. 9I). Notably, we observed that the wild-typefruit grew less after theMGBG treatment, suggesting thatan appropriate level of PAs is essential for fruit cell pro-liferation (Fig. 9, G and H). These results provide strongsupport for the hypothesis that SF2 facilitates cell prolif-eration through modulation of PA homeostasis via tar-geting of SAMDC genes.Finally, we investigated the combined effect of TDZ

and MGBG by spraying female flowers with a mixtureof the chemicals and observed that the treatmentlargely complemented the short-fruit phenotype of sf2,although the transverse diameter also increased (Fig. 9,J and K). We found that the elongation was mostly due

to increased cell numbers (Fig. 9L). Although the wild-type fruit, especially the fruit neck, becamemuch longerafter the treatment (Fig. 9J), the combined effect of TDZand MGBG on sf2 fruit, which resulted in an 80% in-crease in length, was greater than on wild-type fruit,which increased by 16% (Fig. 9K). These results indi-cated that the combined effect of TDZ and MGBG wasmore than additive, suggesting agonistic effects of CKsand PAs on rapid cell proliferation regulated by SF2.Moreover, we observed the fruit-length phenotypes ofthe treated fruit at 16 DAA and found a partial com-plementation of the short-fruit phenotype of sf2, whichincreased in length by 18%, while wild-type fruitshowed no significant change in length compared tountreated plants (Fig. 9, M and N).

DISCUSSION

Evidence of a Direct Role for HDC1 in Cell Proliferation

The basis of size control of multicellular organisms isa longstanding biological question. Two main processes,

Figure 8. Expression patterns of the di-rect SF2 targets in regions undergoingcell proliferation during fruit develop-ment. A, Heat map showing transcriptvariation of core repressed genes duringfruit development from 23 to 8 DAA. B,The RNA level of six core repressedgenes inwild-type (WT) a and sf2 fruits at23, 0, 1, 3, 5, and 8 DAA. UBQ wasused as internal control. C, Heat mapshowing transcript variation of core ac-tivated genes during fruit developmentfrom 23 to 8 DAA. D, The RNA level ofsix core activated genes in WT and sf2fruits at23, 0, 1, 3, 5, and 8 DAA. UBQwas used as internal control. Bars 5means 6 SE of three replicates. **P ,0.05; ***P , 0.01 (t test, one tail).





cell division and cell expansion, underlie final organsize. At the cellular level, cell division is regulated by acombination of two factors: the cell division rate and thecell division duration. Both factors influence the totalnumber of cells in tissues. For instance, in Arabidopsis,transcription factors TEOSINTE BRANCHED1,CYCLOIDEA, and PCF1, and the growth-regulatingfactor are involved in increasing the cell division rate(Powell and Lenhard, 2012). Conversely, the ubiquitin-binding protein DA1 (Li et al., 2008), and theE3 ubiquitin-ligases DA2 and BIG BROTHER (Xia et al.,2013), limit organ size by repressing cell division

duration. In tomato (Solanum lycopersicum), severalgenes have been identified that regulate fruit size andshape by affecting cell division, but the molecularmechanism remains elusive (Frary et al., 2000; Liu et al.,2002; Tanksley, 2004;Wu et al., 2011; Zhang et al., 2012).In cucumber, only a few genes affecting the cell divisionrate or duration have been identified and clonedto date.

Here, we found a recessive allelic variation at SF2,which is homologous to AtHDC1. Both the cell divisionrate and duration were inhibited in fruits of sf2mutant,resulting in 70% reduction in cell numbers in the

Figure 9. SF2 facilitates cell proliferation through direct targeting of CK and PA metabolism and biosynthesis. A, CKX enzymeactivity in 0-DAAwild-type (WT) and sf2 fruits. Bars5mean6 SE of three replicates. B, Endogenous CK content in 0-DAAWTandsf2 fruits. Bars5mean6 SE of three replicates. C to E, Phenotypes and cell size of 0-DAAwild-type and sf2 fruits treatedwithwateror 1mg/L of TDZ. F, Endogenous PA content in 0-DAAWTand sf2 fruits. Bars aremeans6 SE of three replicates. G to I, Phenotypesand cell size of 0-DAAWTand sf2 fruits treatedwithwater or 1mM ofMGBG. J to L, Phenotypes and cell size of 0-DAAWTand sf2fruits treated with water or 1 mg/L of TDZ plus 1 mM of MGBG. M and N, Phenotypes of 16-DAAwild-type and sf2 fruits treatedwith water or 1mg/L of TDZ plus 1mM ofMGBG at the23DAA stage. **P, 0.05; ***P, 0.01 (t test, one tail). Scale bars5 1 cm(C, G, and J), 5 cm (M), and 50 mm (E, I, and L).


Zhang et al.



longitudinal direction of the fruit and a short-fruitphenotype. In contrast to the ubiquitous expressionpattern of SF2 mRNA in various plant tissues, ourstudy showed that the SF2 protein was specificallyexpressed in meristematic tissues in which rapid cellproliferation was occurring (Fig. 3D). A knock-out ofSF2 using CRISPR-Cas9 caused substantial inhibition ofshoot growth (Fig. 2, A–E), consistent with HDC1having a general function in the control of meristematiccell proliferation (Fig. 10A). We inferred that cellnumber represents a major determinant of fruit size,because only fruit length was significantly affected bythe weak allele of sf2 (Fig. 1, A–D). In addition, becausethe G515E mutation in sf2 is located in the yeast regu-lator of transcription3 domain, which is conservedamong angiosperms (Fig. 1N), the function of thismutation is expected to be conserved in plants.HDAC proteins form various types of complexes

through interactions with different regulatory subunits.Our findings indicate that HDC1 is such a regulatorysubunit, and is associated with the site- or tissue-specific function of HDAC in cell proliferation regula-tion. We describe here an elaborate regulatory cellproliferation network, in which SF2 directly targets andrepresses the expression of genes in multiple phyto-hormone pathways, and activates the expression ofgenes involved in cell cycle regulation (Fig. 10B). Itwould be interesting to investigate the interaction pat-tern and regulatory targets of HDC1 in other apical

meristem cells to uncover the core mechanism of reg-ulation of plant cell division.

The HDC1 Regulatory Mechanism Involving HistoneDeacetylation and Gene Regulation

Similar to the results of the genome-wide studies ofHDAC proteins in humans and plants (Wang et al.,2009; Chen et al., 2016a; Yang et al., 2016), SF2, thecucumber HDC1 homolog, mainly targeted geneswith high transcription levels (Fig. 4C) and highacetylation levels (Fig. 5, A and B). This suggests aconserved action of HDAC complexes in regulatingactive genes in eukaryotes. Consistent with the gen-eral function of HDACs proteins as transcriptionalrepressors, we found that the SF2-HDAC complexacts as a direct repressor of target gene networks,including those that suppress auxin, GA, and CK bi-osynthesis and responses, and genes that promote JA,ABA, and PA biosynthesis and responses.Another notable outcome of this study was the

identification of a target gene network that requiresHDC1 directly for its transcriptional activation (Fig. 7;Supplemental Fig. S15). These genes are involved in cellcycle regulation and in phytohormone biosynthesis orsignaling pathways. We showed that the SF2-activatedgenes have higher SF2 binding levels and histoneacetylation levels in the gene body regions, and

Figure 10. Model of SF2/HDC1-mediated cellproliferation. A, The HDAC subunit, SF2/HDC1 isspecifically expressed in meristematic tissues un-dergoing cell proliferation, and has a generalfunction in control of meristematic cell prolifera-tion. Red arrows represent accumulation of HDC1protein causing induction of the function of theHDAC complex in cell proliferation. B, HDACcomplexes consist of different regulatory subunits,such as specific HDAC proteins and transcriptionfactors. SF2/HDC1 recruits theHDAC complex andbinds to the target genes to promote histonedeacetylation and suppress gene expression. TheHDAC complex may also be recruited by SF2 toactivate genes and upregulate their expression bysustaining a dynamic cycle of acetylation anddeacetylation of target genes.






HDC1–HDAC complex is shown to be required forlimiting acetylation in gene bodies (Supplemental Fig.S15). We propose that HDC1 may utilize the samemechanism as that suggested for mammalian HDACs(Zupkovitz et al., 2006; Wang et al., 2009), where HDC1recruits an HDAC complex to active genes, to limitacetylation in gene bodies, and positively regulatetranscription through elongation machinery (Greeret al., 2015). In this scenario, histone deacetylation isessential for keeping transcription at steady-state levels(Wang et al., 2002, 2009). However, there may be an-other regulatory layer of histone deacetylation ontranscriptional activation that is presently not well un-derstood. Our findings indicate that HDC1 employstwo differentmechanisms for regulating transcriptionalrepression and activation during fruit cell proliferation(Fig. 10B).

The complementation of the fruit length phenotypeof sf2 by exogenous treatment with hormones con-firmed the role of SF2 in coordinating hormone bio-synthetic and signaling pathways to facilitate cellproliferation (Fig. 9). An increasing number of studiessuggest a tight link between epigenetic regulation andplant hormone signaling. Several critical regulatoryfactors, such as the chromatin remodeling factor,PICKLE (Ogas et al., 1997), and the corepressor ofHDAC complex, TOPLESS (Pauwels et al., 2010), arepotential key factors in coordinating plant hormonecrosstalk (Yamamuro et al., 2016). Our results point to ageneral role for HDC1 in coordinating phytohormonesignaling through integration with the HDAC complexduring cell proliferation of meristematic cells.

MATERIALS AND METHODS

Plant Materials and Mutant Identification

The cucumber (Cucumis sativus) inbred line 406 was used to make a mutantlibrary, by treating its seeds with 1 mM ethyl methanesulfonate (cat. no. M0880;Sigma-Aldrich) diluted with 0.1 M of P buffer (pH 7.0; Chen et al., 2016b). Theplants of the first mutant generation (M1) were self-pollinated and the sf2mutant was identified in the M2 population. An F2 population was generatedby crossing sf2withwild type 406.Whole genome resequencingwas carried outas reported in Xu et al. (2017). Analysis of the resequencing data revealed 1,023SNPs between the two bulked populations. SNP-index graphs were then cal-culated using these SNPs. We focused on the SNP-index peak on chromosome2, which had an average D(SNP-index). 0.5 and had greatest density of SNPs.Linkage analysis delimited the sf2 locus to a 474-kb interval between two SNPs(2G15049360 and 2G15523545) in the candidate region, and only one SNP(2G15231244) cosegregated with the sf2 locus.

Constructs and Generation of Transgenic Plants

For complementation of sf2, the SF2 coding region together with 995 bp ofpromoter was amplified and cloned into pCAMBIA1300 (Rao et al., 2015;primers are listed in Supplemental Data Set S25). To obtain the SF2 CRISPR/Cas9-edited plants, the binary pBSE402 vector containing a CRISPR cassettewith a functional Cas9 under a constitutive promoter (CaMV 35S) plus a 35S-GFP expression cassette was modified from pBSE401 (a gift from Qijun Chen,China Agricultural University). The single guide RNA (sgRNA) target site fromthe N terminus of SF2was selected (primers are listed in Supplemental Data SetS25). The binary vector pCAMBIA-SF2 or the pBSE402-sgRNA-SF2 was thentransformed into Agrobacterium tumefaciens strain EHA105 by the freeze-thawtransformation protocol (Höfgen and Willmitzer, 1988).

Agrobacterium cells harboring the pCAMBIA-SF2 or the pBSE402-sgRNA-SF2 construct were used to transform the cucumber sf2 or CU2 lines, respec-tively, as described previously by Hu et al. (2017). Cucumber seeds weresterilized and spread on 13 Murashige and Skoog medium (Phytotech) sup-plemented with 2 mg/L of 6-benzylaminopurine (Sigma-Aldrich) and 1 mg/Lof ABA (Phytotech) and left for 2 d at 28°C. Shoot regeneration, elongation, androoting were performed as described previously by Hu et al. (2017).

Genomic DNAwas extracted from callus and plants using the DNeasy PlantMini Kit (Qiagen). PCR was performed using KOD-FX (Toyobo) and the gene-specific primers listed in Supplemental Data Set S25. PCR products were clonedinto pEASY-Blunt Zero (Transgen Biotech) and the SF2 alleles were identifiedby sequencing.

Verification of the Causative SNP using dCAPS Markers

PCR primers for derived cleaved amplified polymorphic sequence (dCAPS)markers (Supplemental Data Set S25) were designed using the software“dCAPS FINDER 2.0” (http://helix.wustl.edu/dcaps/dcaps.html; Neff et al.,1998). The PCR products were digested with restriction enzyme as described inSupplemental Data Set S25 and subsequently separated by electrophoresis in8% polyacrylamide gels.

Measurements of Cell Area and Number

Samples from28,25,23, 0, 3, 5, 8, and 16 DAA of fruits were fixed in a 70%ethanol, acetic acid, and formaldehyde (90:5:5 by volume) solution. Sections (5-mm–thick) were cut with a scalpel from different parts of the fruit (outer,middle, and inner pericarp) and embedded in paraffin (Merck), and then usedto generate 8-mm–thick sections in both widthwise and longitudinal directionsusing a model no. CM3050S microtome (Leica), before staining withhematoxylin-eosin and imaging (Yu et al., 2001). The cell number (X), cell area(A), and average cell area in a given section was calculated using the softwares“Infinity Capture 6.0” (Lumenera Corporation) and “Image Proplus 5.1”(http://www.mediacy.com/imageproplus; Yang et al., 2013). The area of thewhole-fruit cross section or longitudinal section (A9) was determined by mea-suring the ovary or fruit diameter and using the equation for the area of a circle(for a cross section) or an ellipse (for a longitudinal-section). The cell number inwhole fruit cross sections or longitudinal-sections (X9) was calculated by usingthe equation X/A 5 X9/A9. All the measurements were made at three sites ofeach tissue for three sections from each fruit.

Immunoblotting

Proteins were extracted using protein extraction buffer (20mM OF Tris-HCl atpH 7.5, 150 mM of NaCl, 4 M of Urea, 10% glycerol, 5 mM of dithiothreitol [DTT],1 mM of Phenylmethanesulfonyl fluoride [PMSF], and 13 protease inhibitorcocktail [Roche]) and 20–50-mg protein extract was fractionated on a 12% SDS-PAGE gel. The protein concentration was measured according to the Bradfordmethod (Bradford, 1976). Immunoblotting was conducted as reported in Caiet al. (2018). The antibodies used in this study are listed in Supplemental DataSet S26. The SF2 antibody was obtained from MBL Beijing Biotech. The SF2antibody was raised in rabbit using a synthetic peptide corresponding to aminoacids 50–66 of the SF2 protein sequence, and affinity-purified. An additionalCys was added to the C terminus to improve binding.

Immunolabeling

Whole-mount in situ immunocytochemical protein localization in 0-DAAcucumber fruit was performed as described in Paciorek et al. (2006). Primaryrabbit anti-SF2 antibody was used in a 1:20 dilution with Tris-buffered salineTWEEN-20 (10 mM of Tris HCl at pH 8.0, 150 mM of NaCl, 0.05% TWEEN 20,and 0.5% nonfat dry milk) and secondary Fluorescein-Conjugated Goat anti-Rabbit (ZSGB-BIO; https://www.bioz.com/result/zsgb%20bio/product/ZSGB%20Biotech) antibody in a 1:500 dilution with Tris-buffered salineTWEEN-20. Images were collected using a model no. SP8 Confocal Microscope(Leica).

Transient Expression in Leaves of Nicotiana benthamiana

The coding region of SF2was cloned into a binary vector (pCAMBIA1300) tofuse it with an MYC protein downstream of the 35S promoter, using the In-


Zhang et al.









http://helix.wustl.edu/dcaps/dcaps.html


http://www.mediacy.com/imageproplus



https://www.bioz.com/result/zsgb%20bio/product/ZSGB%20Biotech

https://www.bioz.com/result/zsgb%20bio/product/ZSGB%20Biotech


Fusion Cloning Kit (Clontech). The primers used are listed in SupplementalData Set S25. Agro-infiltration for transient expression in leaves of N. ben-thamianawas carried out as in Ting et al. (2013). The experiments were repeatedindependently at least three times with similar results.

Co-IP and LC-MS/MS

Co-IP was performed using 0 DAA of wild type and sf2 fruits and anti-SF2antibodies, as described in Wendrich et al. (2017), with some modifications.Briefly, 1 mg of the antibody bound to 0.5 mg of fruit protein was coupled toDynabeads Protein A (Invitrogen) in PBS buffer (137 mM of NaCl, 2.7 mM ofKCl, 10 mM of Na2HPO4$12H2O, and 2 mM of KH2PO4). After washing with iPbuffer (25 mM of Tris-HCl at pH 7.4, 150 mM of NaCl, 1% NP40, 5% glycerol,1 mM of DTT, 1 mM of PMSF, and protease inhibitor cocktail), the beads wereresuspended in 13 SDS sample buffer (50 mM of Tris at pH 6.8, 10% [v/v]glycerol, 2% [w/v] SDS, 0.1% Coomassie brilliant blue [G-250], and 2% [v/v]b-mercaptoethanol) and the coimmunoprecipitated proteins were separatedusing 12% SDS-PAGE and stained by the staining buffer (a liter containing100 mL of acetic acid, 400 mL of methanol, 1 g of Coomassie brilliant blue [R-250], and 500mL of water). Each lane of the gel was cut into three parts to avoidthe IgG heavy and light chain, and subsequently used for LC-MS/MS as de-scribed in Nallamilli et al. (2013). For immunoblotting, anti-MYC antibody(MBL Beijing Biotech) or anti-FLAG antibody (Sigma-Aldrich) was used(Supplemental Data Set S26).

LCI Assay

The full-length SF2 and sf2 coding sequenceswere fusedwith the C-terminalfragment of firefly luciferase (Luc) in the pCAMBIA-Cluc vector (35S:CLuc-SF2and 35S:CLuc-sf2); SIN3-LIKE1, SIN3-LIKE3, HDA19A, HDA19B, MSI1, andSAP18were fused with the N-terminal fragment of Luc in the pCAMBIA-Nlucvector (35S:SIN3-LIKE1-NLuc, 35S:SIN3-LIKE3-NLuc, 35S:HDA19A-NLuc,35S:HDA19B-NLuc, 35S:MSI1-NLuc, and 35S:SAP18-NLuc) according to Chenet al. (2008). Primers are shown in Supplemental Data Set S25. Agro-infiltrationfor transient expression inN. benthamiana leaves was carried out as described inTing et al. (2013). Fifty hours after coinfiltration in N. benthamiana leaves, theleaves were sprayed with a luciferin solution (100 mM of luciferin and 0.1%Triton X-100) and images captured with a cooled charge-coupled deviceimaging apparatus (Chen et al., 2008). The LUC activity was measured as de-scribed in Chen et al. (2008). The assays were repeated three times.

RNA-Seq Experiment and GO Term Enrichment Analysis

Total RNA was isolated from 0 DAA of wild-type and sf2 fruits in two bi-ological replicates using a TRIzol kit (Invitrogen), according to the instructionmanual. Total RNA from wild-type fruits at different development stages (23,0, 1, 3, 5, and 8 DAA) was isolated in three biological replicates. Paired-endsequencing libraries with an average insert size of 250–300 bp were preparedaccording to the manufacturer’s instructions (Illumina), and 150-bp paired-endreads were generated using a model no. Hiseq2000 Analyzer (Illumina)

All reads were mapped to the reference genome (Huang et al., 2009) withdefault parameters using the software “HISAT2” (https://ccb.jhu.edu/software/hisat2/manual.shtml; Kim et al., 2015). After alignments, gene ex-pression levels were reported as fragments per kilobase of transcript permillionmapped reads, whichwere calculated by the software “StringTie” (https://ccb.jhu.edu/software/stringtie/; Pertea et al., 2016). Differentially expressed geneswere identified through the package “DESEQ R” (http://bioconductor.org/packages/release/bioc/html/DESeq.html; Anders and Huber, 2010) with thecutoff: P value , 0.05 and fold change . 1.5. GO enrichment analyses wereconducted for both the upregulated and downregulated genes using the soft-ware “TopGO” (Alexa and Rahnenfuhrer, 2010).

RT-qPCR and RT-PCR Analysis

One microgram of RNA was reverse-transcribed into complementary DNAwith FastQuant RT Super Mix (Tiangen) according to the manufacturer’s in-structions, followed by qPCR with SYBR Premix (Roche) using an ABI 7900(Salk; primers are listed in Supplemental Data Set S25). Three independentbiological replicates were used. Relative gene expression was calculated usingthe comparative 22△△Ct method (Livak and Schmittgen, 2001).

SF2 transcript levels in different tissues were analyzed by RT-PCR (Chiuet al., 2006) for 28 PCR cycles at an annealing temperature of 58°C. A cucumberubiquitin gene (UBQ; Csa3G778350) was used as a reference. The primers arelisted in Supplemental Data Set S25.

ChIP Assays

ChIP assays were performed with 0 DAA of wild-type and sf2 fruits es-sentially as described in Zhu et al. (2012). The following antibodies were usedfor ChIP assays: SF2 antibody (two biological replicates), anti-H3K9ac (Abcam;two biological replicates), and anti-H3K14ac (Abcam; two biological replicates;Supplemental Data Set S26). ChIP products were combined and eluted into50 mL of Tris-EDTA buffer for ChIP-Seq (.5 ng DNA). The software “MACS2.1.0” (model-based analysis of ChIP-Seq; http://liulab.dfci.harvard.edu/MACS/index.html) is used to identify ChIP-enriched regions for ChIP-Seq data(Zhang et al., 2008).

ChIP-qPCRwas performed as described in Zhu et al. (2012). The primers arelisted in Supplemental Data Set S25. The qPCR signals derived from the ChIPsamples were normalized to the signals derived from the input DNA controlsample. The value (percentage of input; input %) was calculated using the 22ΔCt

method.

CKX Activity Assay

This assay was based on bleaching of 2,6-dichlorophenolindophenol asdescribed in Galuszka et al. (2007) and Frébort et al. (2002). Approximately 1 gof fresh fruit tissue was ground in extraction buffer (0.2 M of Tris-HCl at pH 8.0,0.3% Triton X-100, and 1 mM of PMSF). Cell debris was removed by centrifu-gation at 19,500g for 10 min. The protein extract was incubated in a reactionmixture (total volume of 0.6 mL in 1.5-mL tube) at 37°C in 100 mM of McIlvainebuffer (pH 6.5; McIlvaine, 1921) with 0.5 mM of 2,6-dichlorophenolindophenoland 0.5 mM of iP (6-[g,g-Dimethylallylamino]) for 6 h. This reaction was stop-ped by the addition of 0.3mL of 40% trichloroacetic acid and 2% 4-aminophenolwas added to the supernatant, and the sample was centrifuged at 19,500g for5 min to remove protein precipitate. The resulting concentration of Schiff basewas determined using the molar absorption coefficient (e352 5 15.2 mM21

cm21; Frébort et al., 2002) and expressed as the amount of cleaved iP per totalprotein and reaction time. The fruit protein concentration was measuredaccording to the Bradford method (Bradford, 1976).

Quantification of Endogenous CK Levels

Each sample contained ;120 mg of 0-DAA fruit tissue for quantification ofendogenous CK levels as previously described by Novák et al. (2003) andRiefler et al. (2006). Three independent biological replicates were analyzed.

PA Measurements

Analysis of PAswas carriedout according tomethodsdescribed inFlores andGalston (1982) and Ge et al. (2006). Each sample contained ;100 mg of 0-DAAfruit tissue for quantification of endogenous PA levels. Three independent bi-ological replicates were analyzed.

Statistical Analyses

Statistical analysis was performed using one-tailed Student’s t tests tocompare the two sample groups.

Data Availability

All data that support the findings within this article are available from thecorresponding author upon request.

Accession Numbers

Cucumber genomic sequence data from this article can be found in theCucurbitGenomicsDatabase (icugi.org)under the followingaccessionnumbers:SF2 (Csa2G337260); CKX7 (Csa4G647490); SAMDC1 (Csa2G036680); SAMDC2(Csa3G271360); SIN3-LIKE1 (Csa5G603960); SIN3-LIKE3 (Csa5G484650); MSI1








https://ccb.jhu.edu/software/hisat2/manual.shtml

https://ccb.jhu.edu/software/hisat2/manual.shtml

https://ccb.jhu.edu/software/stringtie/

https://ccb.jhu.edu/software/stringtie/

http://bioconductor.org/packages/release/bioc/html/DESeq.html

http://bioconductor.org/packages/release/bioc/html/DESeq.html




http://liulab.dfci.harvard.edu/MACS/index.htm

http://liulab.dfci.harvard.edu/MACS/index.htm



(Csa3G127190); HDA19A (Csa6G116140); HDA19B (Csa7G029990); SAP18(Csa3G038150).

Sequence data from this article can be found in the GenBank data librariesunder accession number SRP194253.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Morphological changes in wild type and sf2 dur-ing early fruit development.

Supplemental Figure S2. Identification of two independently comple-mented plants.

Supplemental Figure S3. SF2 interacts with the HDAC complex.

Supplemental Figure S4. Quantitative detection of interactions betweenSF2 and HDAC components in N. benthamiana leaves.

Supplemental Figure S5. Co-IP assays indicating the interaction betweenSF2 and SIN3-LIKE proteins in vivo.

Supplemental Figure S6. Representative target genes bound by SF2.

Supplemental Figure S7. Representative DNA motifs identified in SF2binding sites using MEME (Bailey et al., 2006).

Supplemental Figure S8. Genomic distribution of H3K9ac and H3K14acbinding peaks.

Supplemental Figure S9. Genome-wide comparison of H3K9ac andH3K14ac levels in wild-type and sf2 mutant fruits.

Supplemental Figure S10. GO enrichment analyses of genes regulatedby SF2.

Supplemental Figure S11. GO enrichment analysis of 321 core SF2-repressed genes.

Supplemental Figure S12. Analysis of nine core SF2-repressed genes.

Supplemental Figure S13. Analysis of nine core activated genes of SF2.

Supplemental Figure S14. Responsiveness of core SF2-activated genesto TSA.

Supplemental Figure S15. Analysis of SF2 binding and H3Ac levels be-tween SF2-activated genes and SF2-repressed genes.

Supplementary Data Set S1. Phenotypic measurements of selfed F2 plants.

Supplementary Data Set S2. SNP-index from a MutMap analysis (Abeet al., 2012).

Supplementary Data Set S3. LC-MS/MS identification of proteins immu-noprecipitated from wild-type fruit protein extracts using IgG.

Supplementary Data Set S4. LC-MS/MS identification of proteins immu-noprecipitated from wild-type fruit protein extracts using SF2-antibody.

Supplementary Data Set S5. LC-MS/MS identification of proteins immu-noprecipitated from sf2 fruit protein extract using IgG.

Supplementary Data Set S6. LC-MS/MS identification of proteins immu-noprecipitated from sf2 fruit protein extract using SF2-antibody.

Supplementary Data Set S7. Distribution of SF2 complex peaks.

Supplementary Data Set S8. Peaks corresponding to SF2 binding sites.

Supplementary Data Set S9. Distribution of H3K9ac and H3K14ac peaks.

Supplementary Data Set S10. Hyper-H3K9ac peaks.

Supplementary Data Set S11. Hyper-H3K14ac peaks.

Supplementary Data Set S12. The 1,494 upregulated genes in sf2.

Supplementary Data Set S13. The 1,272 downregulated genes in sf2.

Supplementary Data Set S14. The 321 genes that are both SF2 targets andupregulated in sf2.

Supplementary Data Set S15. Overlap between the 321 core repressedgenes and hyper-H3K9ac-enriched genes in sf2.

Supplementary Data Set S16. Overlap between the 321 core repressedgenes and hyper-H3K14ac-enriched genes in sf2.

Supplementary Data Set S17. Fifty-two genes with both H3K9 and H3K14hyperacetylation among the 321 core repressed genes.

Supplementary Data Set S18. Sixteen hormone biosynthesis and signaltransduction genes from the high-confidence 321 core repressed genesthat were targeted and repressed by SF2.

Supplementary Data Set S19. The 237 genes that are both SF2 target genesand downregulated in sf2.

Supplementary Data Set S20. Overlap between the 237 core activatedgenes and hyper-H3K9ac-enriched genes in sf2.

Supplementary Data Set S21. Overlap between the 237 core activatedgenes and hyper-H3K14ac-enriched genes in sf2.

Supplementary Data Set S22. Forty-three genes among the 237 core acti-vated genes with both H3K9 and H3K14 hyperacetylation.

Supplementary Data Set S23. Expression of 321 core repressed genesfrom 23 to 8 DAA during early fruit development.

Supplementary Data Set S24. Expression of 237 core activated genesfrom 23 to 8 DAA during early fruit development.

Supplementary Data Set S25. The primers used in this study.

Supplementary Data Set S26. The antibodies used in this study.

ACKNOWLEDGMENTS

We thank Zhizhong Gong from China Agricultural University for com-ments on the article, PlantScribe (www.plantscribe.com) for editing this article,and Qing Li from the Chinese Academy of Agricultural Sciences for experimen-tal assistance.

Received May 3, 2019; accepted July 22, 2019; published August 4, 2019.

LITERATURE CITED

Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H,Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, et al (2012) Genomesequencing reveals agronomically important loci in rice using MutMap.Nat Biotechnol 30: 174–178

Alexa A, Rahnenfuhrer J (2010) topGO: Enrichment analysis for geneontology. http://bioconductor.org/packages/release/bioc/html/topGO.html

Anders S, Huber W (2010) Differential expression analysis for sequencecount data. Genome Biol 11: R106

Ando K, Grumet R (2010) Transcriptional profiling of rapidly growingcucumber fruit by 454-pyrosequencing analysis. J Am Soc Hortic Sci 135:291–302

Anwar R, Mattoo AK, Handa AK (2015) Polyamine interactions with planthormones: Crosstalk at several levels. In T Kusano and H Suzuki,, eds,Polyamines: A Universal Molecular Nexus for Growth, Survival, andSpecialized Metabolism. Springer, Tokyo, pp 267–302

Bailey TL, Williams N, Misleh C, Li WW (2006) MEME: Discovering andanalyzing DNA and protein sequence motifs. Nucleic Acids Res 34:W369–W373

Bo K, Ma Z, Chen J, Weng Y (2015) Molecular mapping reveals structuralrearrangements and quantitative trait loci underlying traits with localadaptation in semi-wild Xishuangbanna cucumber (Cucumis sativus L.var. xishuangbannanesis Qi et Yuan). Theor Appl Genet 128: 25–39

Bou-Torrent J, Roig-Villanova I, Galstyan A, Martínez-García JF (2008)PAR1 and PAR2 integrate shade and hormone transcriptional networks.Plant Signal Behav 3: 453–454

Bradford MM (1976) A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 72: 248–254

Cai J, Qin G, Chen T, Tian S (2018) The mode of action of remorin1 inregulating fruit ripening at transcriptional and post-transcriptionallevels. New Phytol 219: 1406–1420


Zhang et al.











































http://www.plantscribe.com

http://bioconductor.org/packages/release/bioc/html/topGO.html

http://bioconductor.org/packages/release/bioc/html/topGO.html


Chen H, Zou Y, Shang Y, Lin H, Wang Y, Cai R, Tang X, Zhou J-M (2008)Firefly luciferase complementation imaging assay for protein–proteininteractions in plants. Plant Physiol 146: 368–376

Chen H, Sun J, Li S, Cui Q, Zhang H, Xin F, Wang H, Lin T, Gao D, WangS, et al (2016b) An ACC oxidase gene essential for cucumber carpeldevelopment. Mol Plant 9: 1315–1327

Chen X, Lu L, Mayer KS, Scalf M, Qian S, Lomax A, Smith LM, Zhong X(2016a) POWERDRESS interacts with HISTONE DEACETYLASE 9 topromote aging in Arabidopsis. eLife 5: e17214

Chiu WB, Lin CH, Chang CJ, Hsieh MH, Wang AY (2006) Molecularcharacterization and expression of four cDNAs encoding sucrose syn-thase from green bamboo Bambusa oldhamii. New Phytol 170: 53–63

Cigliano RA, Cremona G, Paparo R, Termolino P, Perrella G, Gutzat R,Consiglio MF, Conicella C (2013) Histone deacetylase AtHDA7 is re-quired for female gametophyte and embryo development in Arabidopsis.Plant Physiol 163: 431–440

Colle M, Weng Y, Kang Y, Ophir R, Sherman A, Grumet R (2017) Vari-ation in cucumber (Cucumis sativus L.) fruit size and shape results frommultiple components acting pre-anthesis and post-pollination. Planta246: 641–658

de Jager SM, Maughan S, Dewitte W, Scofield S, Murray JA (2005) Thedevelopmental context of cell-cycle control in plants. Semin Cell DevBiol 16: 385–396

Flores HE, Galston AW (1982) Analysis of polyamines in higher plants byhigh performance liquid chromatography. Plant Physiol 69: 701–706

Frary A, Nesbitt TC, Grandillo S, Knaap E, Cong B, Liu J, Meller J, ElberR, Alpert KB, Tanksley SD (2000) fw2.2: A quantitative trait locus keyto the evolution of tomato fruit size. Science 289: 85–88

Frébort I, Šebela M, Galuszka P, Werner T, Schmülling T, Pec P (2002)Cytokinin oxidase/cytokinin dehydrogenase assay: Optimized proce-dures and applications. Anal Biochem 306: 1–7

Friml J, Yang X, Michniewicz M, Weijers D, Quint A, Tietz O, BenjaminsR, Ouwerkerk PB, Ljung K, Sandberg G, et al (2004) A PINOID-dependent binary switch in apical-basal PIN polar targeting directsauxin efflux. Science 306: 862–865

Galuszka P, Popelková H, Werner T, Frébortová J, Pospísilová H, Mik V,Köllmer I, Schmülling T, Frébort I (2007) Biochemical characterizationof cytokinin oxidases/dehydrogenases from Arabidopsis thaliana ex-pressed in Nicotiana tabacum L. J Plant Growth Regul 26: 255–267

Ge C, Cui X, Wang Y, Hu Y, Fu Z, Zhang D, Cheng Z, Li J (2006) BUD2,encoding an S-adenosylmethionine decarboxylase, is required for Ara-bidopsis growth and development. Cell Res 16: 446–456

Girin T, Sorefan K, Østergaard L (2009) Meristematic sculpting in fruitdevelopment. J Exp Bot 60: 1493–1502

Gonzalez D, Bowen AJ, Carroll TS, Conlan RS (2007) The transcriptioncorepressor LEUNIG interacts with the histone deacetylase HDA19 andmediator components MED14 (SWP) and CDK8 (HEN3) to represstranscription. Mol Cell Biol 27: 5306–5315

Greer CB, Tanaka Y, Kim YJ, Xie P, Zhang MQ, Park IH, Kim TH (2015)Histone deacetylases positively regulate transcription through theelongation machinery. Cell Reports 13: 1444–1455

Griffiths J, Murase K, Rieu I, Zentella R, Zhang Z-L, Powers SJ, Gong F,Phillips AL, Hedden P, Sun TP, et al (2006) Genetic characterizationand functional analysis of the GID1 gibberellin receptors in Arabidopsis.Plant Cell 18: 3399–3414

Grumet R, Colle M (2016) Genomic analysis of cucurbit fruit growth. In RGrumet, N Katzir, J Garcia-Mas, eds, Genetics and Genomics of Cu-curbitaceae. Springer, New York, pp 321–344

Grzenda A, Lomberk G, Zhang J-S, Urrutia R (2009) Sin3: Master scaffoldand transcriptional corepressor. Biochim Biophys Acta 1789: 443–450

Hare PD, Van Staden J (1994) Inhibitory effect of thidiazuron on the ac-tivity of cytokinin oxidase isolated from soybean callus. Plant CellPhysiol 35: 1121–1125

Höfgen R, Willmitzer L (1988) Storage of competent cells for Agro-bacterium transformation. Nucleic Acids Res 16: 9877

Hu B, Li D, Liu X, Qi J, Gao D, Zhao S, Huang S, Sun J, Yang L (2017)Engineering non-transgenic gynoecious cucumber using an improvedtransformation protocol and optimized CRISPR/Cas9 system. Mol Plant10: 1575–1578

Huang S, Li R, Zhang Z, Li L, Gu X, Fan W, Lucas WJ, Wang X, Xie B, NiP, et al (2009) The genome of the cucumber, Cucumis sativus L. Nat Genet41: 1275–1281

Ikeuchi M, Iwase A, Sugimoto K (2015) Control of plant cell differentiationby histone modification and DNA methylation. Curr Opin Plant Biol 28:60–67

Jamaladdin S, Kelly RDW, O’Regan L, Dovey OM, Hodson GE, MillardCJ, Portolano N, Fry AM, Schwabe JWR, Cowley SM (2014) Histonedeacetylase (HDAC) 1 and 2 are essential for accurate cell division andthe pluripotency of embryonic stem cells. Proc Natl Acad Sci USA 111:9840–9845

Kaur-Sawhney R, Tiburcio AF, Altabella T, Galston AW (2003) Polya-mines in plants: An overview. J Cell Mol Biol 2: 1–12

Kim D, Langmead B, Salzberg SL (2015) HISAT: A fast spliced alignerwith low memory requirements. Nat Methods 12: 357–360

Kouzarides T (1999) Histone acetylases and deacetylases in cell prolifera-tion. Curr Opin Genet Dev 9: 40–48

Li Y, Zheng L, Corke F, Smith C, Bevan MW (2008) Control of final seedand organ size by the DA1 gene family in Arabidopsis thaliana. Genes Dev22: 1331–1336

Liu J, Van Eck J, Cong B, Tanksley SD (2002) A new class of regulatorygenes underlying the cause of pear-shaped tomato fruit. Proc Natl AcadSci USA 99: 13302–13306

Liu X, Yang S, Zhao M, Luo M, Yu C-W, Chen C-Y, Tai R, Wu K (2014)Transcriptional repression by histone deacetylases in plants. Mol Plant7: 764–772

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression datausing real-time quantitative PCR and the 2(2DDC(T)) method. Methods 25:402–408

Long JA, Ohno C, Smith ZR, Meyerowitz EM (2006) TOPLESS regulatesapical embryonic fate in Arabidopsis. Science 312: 1520–1523

McIlvaine T (1921) A buffer solution for colorimetric comparison.J Biochem 49: 183–186

Murase K, Hirano Y, Sun TP, Hakoshima T (2008) Gibberellin-inducedDELLA recognition by the gibberellin receptor GID1. Nature 456:459–463

Nallamilli BRR, Zhang J, Mujahid H, Malone BM, Bridges SM, Peng Z(2013) Polycomb group gene OsFIE2 regulates rice (Oryza sativa) seeddevelopment and grain filling via a mechanism distinct from Arabidopsis.PLoS Genet 9: e1003322

Neff MM, Neff JD, Chory J, Pepper AE (1998) dCAPS, a simple techniquefor the genetic analysis of single nucleotide polymorphisms: Experi-mental applications in Arabidopsis thaliana genetics. Plant J 14: 387–392

Niu B, Wang L, Zhang L, Ren D, Ren R, Copenhaver GP, Ma H, Wang Y(2015) Arabidopsis Cell Division Cycle 20.1 is required for normal meioticspindle assembly and chromosome segregation. Plant Cell 27: 3367–3382

Novák O, Tarkowski P, Tarkowská D, Dole�zal K, Lenobel R, Strnad M(2003) Quantitative analysis of cytokinins in plants by liquidchromatography–single-quadrupole mass spectrometry. Anal ChimActa 480: 207–218

Ogas J, Cheng J-C, Sung ZR, Somerville C (1997) Cellular differentiationregulated by gibberellin in the Arabidopsis thaliana pickle mutant. Science277: 91–94

Paciorek T, Sauer M, Balla J, Wi�sniewska J, Friml J (2006) Immunocyto-chemical technique for protein localization in sections of plant tissues.Nat Protoc 1: 104–107

Pan Y, Liang X, Gao M, Liu H, Meng H, Weng Y, Cheng Z (2017) Roundfruit shape in WI7239 cucumber is controlled by two interacting quan-titative trait loci with one putatively encoding a tomato SUN homolog.Theor Appl Genet 130: 573–586

Park S-Y, Fung P, Nishimura N, Jensen DR, Fujii H, Zhao Y, Lumba S,Santiago J, Rodrigues A, Chow TF, et al (2009) Abscisic acid inhibitstype 2C protein phosphatases via the PYR/PYL family of START pro-teins. Science 324: 1068–1071

Pastuglia M, Azimzadeh J, Goussot M, Camilleri C, Belcram K, EvrardJ-L, Schmit A-C, Guerche P, Bouchez D (2006) g-tubulin is essential formicrotubule organization and development in Arabidopsis. Plant Cell 18:1412–1425

Pauwels L, Barbero GF, Geerinck J, Tilleman S, Grunewald W, Pérez AC,Chico JM, Bossche RV, Sewell J, Gil E, et al (2010) NINJA connects theco-repressor TOPLESS to jasmonate signalling. Nature 464: 788–791

Pedersen DS, Coppens F, Ma L, Antosch M, Marktl B, Merkle T,Beemster GT, Houben A, Grasser KD (2011) The plant-specific familyof DNA-binding proteins containing three HMG-box domains interactswith mitotic and meiotic chromosomes. New Phytol 192: 577–589





Perrella G, Lopez-Vernaza MA, Carr C, Sani E, Gosselé V, Verduyn C,Kellermeier F, Hannah MA, Amtmann A (2013) Histone deacetylasecomplex1 expression level titrates plant growth and abscisic acid sen-sitivity in Arabidopsis. Plant Cell 25: 3491–3505

Perrella G, Carr C, Asensi-Fabado MA, Donald NA, Páldi K, Hannah MA,Amtmann A (2016) The Histone Deacetylase Complex (HDC) 1 proteinof Arabidopsis thaliana has the capacity to interact with multiple proteinsincluding histone 3-binding proteins and histone 1 variants. PlantPhysiol 171: 62–70

Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-levelexpression analysis of RNA-seq experiments with HISAT, StringTie andBallgown. Nat Protoc 11: 1650–1667

Powell AE, Lenhard M (2012) Control of organ size in plants. Curr Biol 22:R360–R367

Qi J, Liu X, Shen D, Miao H, Xie B, Li X, Zeng P, Wang S, Shang Y, Gu X,et al (2013) A genomic variation map provides insights into the geneticbasis of cucumber domestication and diversity. Nat Genet 45: 1510–1515

Rao Y, Yang Y, Xu J, Li X, Leng Y, Dai L, Huang L, Shao G, Ren D, Hu J,et al (2015) EARLY SENESCENCE1 encodes a SCAR-LIKE PROTEIN2that affects water loss in rice. Plant Physiol 169: 1225–1239

Riefler M, Novak O, Strnad M, Schmülling T (2006) Arabidopsis cytokininreceptor mutants reveal functions in shoot growth, leaf senescence, seedsize, germination, root development, and cytokinin metabolism. PlantCell 18: 40–54

Rossi V, Locatelli S, Varotto S, Donn G, Pirona R, Henderson DA,Hartings H, Motto M (2007) Maize histone deacetylase hda101 is in-volved in plant development, gene transcription, and sequence-specificmodulation of histone modification of genes and repeats. Plant Cell 19:1145–1162

Shahbazian MD, Grunstein M (2007) Functions of site-specific histoneacetylation and deacetylation. Annu Rev Biochem 76: 75–100

Tanksley SD (2004) The genetic, developmental, and molecular bases offruit size and shape variation in tomato. Plant Cell 16(Suppl): S181–S189

Tian L, Chen ZJ (2001) Blocking histone deacetylation in Arabidopsis in-duces pleiotropic effects on plant gene regulation and development.Proc Natl Acad Sci USA 98: 200–205

Tian L, Fong MP, Wang JJ, Wei NE, Jiang H, Doerge RW, Chen ZJ (2005)Reversible histone acetylation and deacetylation mediate genome-wide,promoter-dependent and locus-specific changes in gene expressionduring plant development. Genetics 169: 337–345

Ting HM, Wang B, Rydén AM, Woittiez L, van Herpen T, VerstappenFW, Ruyter-Spira C, Beekwilder J, Bouwmeester HJ, van der Krol A(2013) The metabolite chemotype of Nicotiana benthamiana transientlyexpressing artemisinin biosynthetic pathway genes is a function ofCYP71AV1 type and relative gene dosage. New Phytol 199: 352–366

Tokunaga H, Kojima M, Kuroha T, Ishida T, Sugimoto K, Kiba T,Sakakibara H (2012) Arabidopsis lonely guy (LOG) multiple mutantsreveal a central role of the LOG-dependent pathway in cytokinin acti-vation. Plant J 69: 355–365

Trivedi CM, Zhu W, Wang Q, Jia C, Kee HJ, Li L, Hannenhalli S, EpsteinJA (2010) Hopx and Hdac2 interact to modulate Gata4 acetylation andembryonic cardiac myocyte proliferation. Dev Cell 19: 450–459

Ueno Y, Ishikawa T, Watanabe K, Terakura S, Iwakawa H, Okada K,Machida C, Machida Y (2007) Histone deacetylases and ASYMMETRICLEAVES2 are involved in the establishment of polarity in leaves ofArabidopsis. Plant Cell 19: 445–457

Utley RT, Ikeda K, Grant PA, Côté J, Steger DJ, Eberharter A, John S,Workman JL (1998) Transcriptional activators direct histone acetyl-transferase complexes to nucleosomes. Nature 394: 498–502

Wang A, Kurdistani SK, Grunstein M (2002) Requirement of Hos2 histonedeacetylase for gene activity in yeast. Science 298: 1412–1414

Wang Z, Zang C, Cui K, Schones DE, Barski A, Peng W, Zhao K (2009)Genome-wide mapping of HATs and HDACs reveals distinct functionsin active and inactive genes. Cell 138: 1019–1031

Wei Q, Wang Y, Qin X, Zhang Y, Zhang Z, Wang J, Li J, Lou Q, Chen J(2014) An SNP-based saturated genetic map and QTL analysis of fruit-related traits in cucumber using specific-length amplified fragment(SLAF) sequencing. BMC Genomics 15: 1158

Wendrich JR, Boeren S, Möller BK, Weijers D, De Rybel B (2017) Invivo identification of plant protein complexes using IP-MS/MS. InJ Kleine-Vehn and M Sauer,, eds, Plant Hormones. Methods in Molec-ular Biology, Vol 1497. Humana Press, New York, pp 147–158

Weng Y, Colle M, Wang Y, Yang L, Rubinstein M, Sherman A, Ophir R,Grumet R (2015) QTL mapping in multiple populations and develop-ment stages reveals dynamic quantitative trait loci for fruit size in cu-cumbers of different market classes. Theor Appl Genet 128: 1747–1763

Werner T, Motyka V, Strnad M, Schmülling T (2001) Regulation of plantgrowth by cytokinin. Proc Natl Acad Sci USA 98: 10487–10492

Wu S, Xiao H, Cabrera A, Meulia T, van der Knaap E (2011) SUN regulatesvegetative and reproductive organ shape by changing cell divisionpatterns. Plant Physiol 157: 1175–1186

Xia T, Li N, Dumenil J, Li J, Kamenski A, Bevan MW, Gao F, Li Y (2013)The ubiquitin receptor DA1 interacts with the E3 ubiquitin ligase DA2 toregulate seed and organ size in Arabidopsis. Plant Cell 25: 3347–3359

Xiong L, Lee H, Ishitani M, Zhu J-K (2002) Regulation of osmotic stress-responsive gene expression by the LOS6/ABA1 locus in Arabidopsis.J Biol Chem 277: 8588–8596

Xu J, Shi S, Wang L, Tang Z, Lv T, Zhu X, Ding X, Wang Y, Zhao FJ, Wu Z(2017) OsHAC4 is critical for arsenate tolerance and regulates arsenicaccumulation in rice. New Phytol 215: 1090–1101

Yamamuro C, Zhu J-K, Yang Z (2016) Epigenetic modifications and planthormone action. Mol Plant 9: 57–70

Yang H, Liu X, Xin M, Du J, Hu Z, Peng H, Rossi V, Sun Q, Ni Z, Yao Y(2016) Genome-wide mapping of targets of maize histone deacetylaseHDA101 reveals its function and regulatory mechanism during seeddevelopment. Plant Cell 28: 629–645

Yang XY, Wang Y, Jiang WJ, Liu XL, Zhang XM, Yu HJ, Huang SW, LiuGQ (2013) Characterization and expression profiling of cucumber ki-nesin genes during early fruit development: Revealing the roles of ki-nesins in exponential cell production and enlargement in cucumberfruit. J Exp Bot 64: 4541–4557

Yoshida R, Umezawa T, Mizoguchi T, Takahashi S, Takahashi F,Shinozaki K (2006) The regulatory domain of SRK2E/OST1/SnRK2.6interacts with ABI1 and integrates abscisic acid (ABA) and osmoticstress signals controlling stomatal closure in Arabidopsis. J Biol Chem281: 5310–5318

Yu JQ, Li Y, Qian YR, Zhu ZJ (2001) Cell division and cell enlargement infruit of Lagenaria leucantha as influenced by pollination and plant growthsubstances. Plant Growth Regul 33: 117–122

Zhang F, Wang L, Ko EE, Shao K, Qiao H (2018) Histone deacetylasesSRT1 and SRT2 interact with ENAP1 to mediate ethylene-inducedtranscriptional repression. Plant Cell 30: 153–166

Zhang N, Brewer MT, van der Knaap E (2012) Fine mapping of fw3.2controlling fruit weight in tomato. Theor Appl Genet 125: 273–284

Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE,Nusbaum C, Myers RM, Brown M, Li W, et al (2008) Model-basedanalysis of ChIP-Seq (MACS). Genome Biol 9: R137

Zhu JY, Sun Y, Wang Z-Y (2012) Genome-wide identification of tran-scription factor-binding sites in plants using chromatin immunopre-cipitation followed by microarray (ChIP-chip) or sequencing (ChIP-seq).Methods Mol Biol 876: 173–188

Zilio N, Codlin S, Vashisht AA, Bitton DA, Head SR, Wohlschlegel JA,Bähler J, Boddy MN (2014) A novel histone deacetylase complex in thecontrol of transcription and genome stability. Mol Cell Biol 34:3500–3514

Zupkovitz G, Tischler J, Posch M, Sadzak I, Ramsauer K, Egger G,Grausenburger R, Schweifer N, Chiocca S, Decker T, et al (2006)Negative and positive regulation of gene expression by mouse histonedeacetylase 1. Mol Cell Biol 26: 7913–7928


Zhang et al.



genome-wide target mapping shows histone deacetylase ... · genome-wide target mapping shows...

Documents