LARGE-SCALE BIOLOGY ARTICLE

Transcriptomics at Maize Embryo/Endosperm InterfacesIdentifies a Transcriptionally Distinct Endosperm SubdomainAdjacent to the Embryo Scutellum[OPEN]

Nicolas M. Doll,a Jeremy Just,a Véronique Brunaud,b,c José Caïus,b,c Aurélie Grimault,a Nathalie Depège-Fargeix,a

Eddi Esteban,d Asher Pasha,d Nicholas J. Provart,d Gwyneth C. Ingram,a Peter M. Rogowsky,a andThomas Widieza,1

a Laboratoire Reproduction et Developpement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRAE, F-69342 Lyon,Franceb Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université Evry, Université Paris-Saclay, F-91405Orsay, Francec Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, F-91405 Orsay, FrancedDepartment of Cell and Systems Biology/Centre for the Analysis of Genome Evolution and Function, University of Toronto, Toronto,Ontario M5S 3B2, Canada

ORCID IDs: 0000-0001-5429-8920 (N.M.D.); 0000-0003-0842-9808 (J.J.); 0000-0002-6246-3161 (V.B.); 0000-0001-8692-042X (J.C.);0000-0002-7957-7288 (A.G.); 0000-0003-4591-1791 (N.D.-F.); 0000-0001-9016-9202 (E.E.); 0000-0002-9315-0520 (A.P.); 0000-0001-5551-7232 (N.J.P.); 0000-0002-1425-9545 (G.C.I.); 0000-0003-4822-3783 (P.M.R.); 0000-0001-6002-2306 (T.W.)

Seeds are complex biological systems comprising three genetically distinct tissues nested one inside another (embryo,endosperm, and maternal tissues). However, the complexity of the kernel makes it difficult to understand intercompartmentinteractions without access to spatially accurate information. Here, we took advantage of the large size of the maize (Zeamays) kernel to characterize genome-wide expression profiles of tissues at different embryo/endosperm interfaces. Ouranalysis identifies specific transcriptomic signatures in two interface tissues compared with whole seed compartments: thescutellar aleurone layer and the newly named endosperm adjacent to scutellum (EAS). The EAS, which appears around 9 dafter pollination and persists for around 11 d, is confined to one to three endosperm cell layers adjacent to the embryonicscutellum. Its transcriptome is enriched in genes encoding transporters. The absence of the embryo in an embryo specificmutant can alter the expression pattern of EAS marker genes. The detection of cell death in some EAS cells together with anaccumulation of crushed cell walls suggests that the EAS is a dynamic zone from which cell layers in contact with the embryoare regularly eliminated and to which additional endosperm cells are recruited as the embryo grows.

INTRODUCTION

Cereal grains are not only essential for plant propagation but arealso high-value products that represent an important source ofcalories and proteins for human nutrition and animal feed as wellas a coveted resource for bio-sourced industries. In maize (Zeamays), the accumulation of oil in the embryo and of starch andprotein in the endosperm requires the development of adaptedstructures and the coordinated regulation and distribution ofnutrient flow from the mother plant. The development of theembryo, which will form the future plant, and the endosperm,which will nourish the embryo during germination, occurs in three

main phases (Lopes and Larkins, 1993; Berger, 1999; Dumas andRogowsky, 2008). During the first 2 weeks of early maize seeddevelopment, embryo and endosperm cells differentiate intopopulations forming distinct tissues and organs (Randolph, 1936;Lerouxet al., 2014), including twostorageorgans, the scutellumofthe embryo and the starchy endosperm (early developmentphase). These twozygoticcompartments thenstart toaccumulatelarge quantities of storage compounds during the following 2 to3 weeks (filling phase), while the surrounding maternal tissuesprovide or transport the necessary nutrient supplies (Porter et al.,1987;WuandMessing,2014).During thefinal4weeks (maturationphase), the kernel dehydrates and enters into quiescence priorto dispersal (Vernoud et al., 2005; Sabelli and Larkins, 2009;Sreenivasulu and Wobus, 2013). These three phases are de-termined by distinct genetic programs and characterized bydistinct anatomical and cytological features. Spatially, the maizekernel is organized like Russian dolls, the embryo being enclosedwithin the endosperm, which is itself surrounded by the pericarp(maternal tissues).

1 Address correspondence to thomas.widiez@ens-lyon.fr.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: Thomas Widiez (thomas.widiez@ens-lyon.fr)[OPEN]Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.19.00756

The Plant Cell, Vol. 32: 833–852, April 2020, www.plantcell.org ã 2020 ASPB.

A closer look at the highly differentiated structure displayed bythe maize embryo shows that 4 d after pollination (DAP), twodistinct parts can be distinguished: an apical embryo proper anda basal suspensor that will degenerate at the end of early de-velopment (Giuliani et al., 2002; Doll et al., 2017). At around 8DAP,the embryo proper generates, at the abaxial side, a shield-shapedorgan, the above-mentioned scutellum. The shoot apical meri-stem develops on the adaxial side. Marking the apical pole of thefuture embryonic axis, the shoot apical meristem will produceseveral embryonic leaves over time. The root apical meristemdifferentiates within the embryo body, defining the basal pole ofthe embryonic axis. Shoot and root meristems will be surroundedby theprotectivecoleoptile andcoleorhiza, respectively (Randolph,1936; Bommert and Werr, 2001; Vernoud et al., 2005).

The surrounding endosperm,which occupies 70%of the kernelvolumeat the endof early development (Sabelli andLarkins, 2009;Leroux et al., 2014; Rousseau et al., 2015; Zhan et al., 2017), hasbeen described as differentiating only four main cell types. Thebasal endosperm transfer layer (BETL) and the aleurone layer (AL)are two peripheral cell types in contact with maternal tissues. Theembryo-surrounding region (ESR) is formed of small denselycytoplasmic cells encircling the young embryo. Lastly, the starchyendosperm (SE) corresponds to the central region of the endo-sperm, which subsequently accumulates huge amounts of stor-age compounds before undergoing progressive programmed celldeath. The developing endosperm is surrounded by maternaltissues: the nutritive nucellus, which degenerates as the endo-sperm expands, and the protective pericarp, which comprises thepedicel at the basal pole (Olsen, 2001; Berger, 2003; Sabelli andLarkins, 2009; Zhan et al., 2017).

The parallel growth and profound developmental trans-formations of the three kernel compartments highlight the needfor constant coordination, which likely requires a complex

intercompartmental dialogue (Nowack et al., 2010; Ingram andGutierrez-Marcos, 2015; Widiez et al., 2017). Since maternaltissues, endosperm, and embryo are symplastically isolated, theirapoplastic interfaces represent essential zones for this dialogue(Diboll and Larson, 1966; Van Lammeren, 1987; Widiez et al.,2017). A good example to illustrate the importance and special-ization of interfaces is carbon transport. Sugars must be trans-ported from the maternal tissues to the embryo for growth andfatty acid accumulation, passing through the endosperm, whichneeds to retain part of the carbon for its own growth as well asthe biosynthesis of starch and storage proteins (Sabelli andLarkins, 2009; Chourey and Hueros, 2017). In maize, nutrientsare unloaded from open ends of the phloem vessels into theplacento-chalazal zoneof thematernal pedicel (Porter et al., 1987;Bezrutczyk et al., 2018). At the base of the endosperm, the BETLcells form dramatic cell wall ingrowths, thus increasing the ex-changesurface (KiesselbachandWalker, 1952;Davis et al., 1990).BETL cells express a specific set of genes, including Miniature1,encodingacellwall invertase,whichcleavessucrose intohexoses(Lowe and Nelson, 1946; Miller and Chourey, 1992; Cheng et al.,1996; Kang et al., 2009). These are taken up by the sugartransporter SWEET4c (Sugars Will Eventually be ExportedTransporter), which has been demonstrated to be the keytransporter of sugar at the pedicel/endosperm interface, since thedefects in seedfillingof the corresponding sweet4cmutant lead toa miniature kernel phenotype (Sosso et al., 2015). The remainingendosperm interface with maternal tissues (initially the nucellusand later on the pericarp) is the AL, which is not known to con-tribute to nutrient exchange during seed development (Gontarekand Becraft, 2017).The interface between the endosperm and the embryo is also

developmentally dynamic. At 3 to 6 DAP, the embryo is totallysurroundedbyESR-typecells.As theembryoexpands, it emerges

834 The Plant Cell

from the ESR, which consequently becomes restricted to thezone surrounding the basal part (suspensor) of the embryo andultimately disappears together with the suspensor at the end ofthe early development phase (Opsahl-Ferstad et al., 1997;Giuliani et al., 2002). From 8 to 9 DAP, the upper part (embryoproper) forms two new interfaces: (1) at the adaxial side, theembryo is enclosed by a single cell layer, which is called thescutellar aleurone layer (SAL) in barley (Hordeumvulgare; Jestinet al., 2008); and (2) at the abaxial side, the embryo is broughtinto direct contact with central SE cells (Van Lammeren, 1987).This interface is constantly moving due to the growth of thescutellum inside the endosperm. On the embryo side of thisinterface, the epidermis of the scutellum has a distinct mor-phology and gene expression pattern (Ingram et al., 2000;Bommert and Werr, 2001). The dynamics of the endosperm/embryo interface, and the processes that occur there, remainlargely undescribed.

At many intercompartmental interfaces, such as the BETL, theESR, and the AL, the cells constitute readily identifiable tissueswith distinctive and often striking cell morphologies and withdefined organizations and established functions (except for theESR; for review, see Doll et al., 2017). Inmany cases, specific setsof genes are expressed in these tissues, as revealed by theidentificationandcharacterizationofmarker genes, for exampleofMaternally expressed gene1, Myb-related protein1, and Betl1,Betl2, Betl3, and Betl4 in the BETL (Hueros et al., 1999a, 1999b;Cai et al., 2002;Gómezet al., 2002;Gutiérrez-Marcos et al., 2004),Viviparous1 in the AL (Suzuki et al., 2003), and Esr1 to Esr3 in theESR (Opsahl-Ferstad et al., 1997).

Genome-wide gene expression studies at numerous de-velopmental stages of whole kernels and/or hand-dissectedendosperm and embryo (Downs et al., 2013; Lu et al., 2013; Chenet al., 2014; Li et al., 2014; Qu et al., 2016; Meng et al., 2018) havebeen complemented by a recent transcriptomic analysis oflaser-capturemicrodissected cell types and subcompartmentsof 8-DAP kernels (Zhan et al., 2015). However, even the latterstudy did not address specifically the transcriptomic profiles ofthe embryo/endosperm interfaces and did not answer thequestion of whether the endosperm at the scutellum/endosperminterface is composed of cells with specific transcriptionalidentities.

In this study, we took advantage of the large size of the maizekernel to characterize thegenome-widegeneexpressionprofile atembryo/endosperm interfaces at 13 DAP. RNA-seq profiling re-vealed that endosperm cells in close contact with the embryoscutellum have a distinct transcriptional signature, allowing us todefine an endosperm zone we named the EAS for endospermadjacent to scutellum, which is specialized in nutrient transportbased on Gene Ontology (GO) enrichment analysis. In situ hy-bridization shows that the EAS is confined to one to three en-dosperm cell layers adjacent to the scutellum, whereas kineticanalyses show that the EAS is present when the scutellumemergesataround9DAPandpersists throughoutembryogrowth,up to;20DAP.Thedetectionofcell death in theEAStogetherwiththe impaired expression of EAS marker genes in an embryospecific mutant suggest that the EAS is a developmentally dy-namic interface influenced by the presence of the neighboringgrowing embryo.

RESULTS

RNA-Seq Profiling of 13-DAP Maize Kernel Compartmentsand Embryo/Endosperm Interfaces

To obtain the gene expression patterns of embryo/endosperminterfaces in maize kernels, six (sub)compartments were hand-dissected for transcriptomic analysis (Figure 1; SupplementalFigure 1). The three whole compartments were the maternaltissues excluding the pedicel, which were labeled pericarp (Per),the whole endosperm (End), and the whole embryo (Emb; Fig-ure 1). The subcompartments corresponding to three distinctembryo/endosperm interfaces were the SAL (the single endo-sperm cell layer at the adaxial side of the embryo), the apicalscutellum (AS; corresponding to the embryo tip composeduniquely of scutellum tissues without the embryo axis), anda new region that we named the EAS, corresponding to severallayers of endosperm cells in close contact with the scutellum atthe abaxial side of the embryo (Figure 1; Supplemental Figure 1).The tissues were collected from kernels of inbred line B73 (usedto establish the maize reference genome) at 13 DAP (embryosize of ;2.5 mm), the earliest developmental stage at whichhand dissection of these embryo/endosperm interfaces wasfeasible, and also the transition from early development to thefilling phase.For each of the six samples, four biological replicates, each

composed of a pool of dissected tissues from two differentplants,wereproduced (Supplemental Table 1). A total of 24RNA-seq libraries were constructed and sequenced in paired-endmode using Illumina HiSeq2000 technology. The resulting reads(on average 62 million pairs per sample) were checked for quality,cleaned, and mapped to the current version of the B73 maizereference genome (AGP v4). On average, 95.8% 6 0.4% of thepairs were mapped, and on average, 78.3% 6 5.3% corre-sponded to annotated genes (Supplemental Figure 2A). Pairsthat mapped to multiple genes (10.2% 6 5.3%) or to no gene(5.2% 6 1.1%), as well as ambiguous hits (1.5% 6 0.6%), werefiltered (Supplemental Figure2A).Agenewasconsidered tobenotexpressed if it gives rise to less than one read per million. At least25,000 genes were found to be expressed per replicate, with thelargest number found in the SAL (;30,000 genes expressed;Supplemental Figure 2B). The results generated for each replicateare available in Supplemental Data Set 1. Venn diagrams weregenerated to visualize overlaps between the sets of genes ex-pressed in the threewhole compartments (Per, Emb, andEnd) andbetween the sets of genes expressed in the End and the twoendosperm subcompartments (EASandSAL; Figures 2Aand 2B).In order to assess the relationships between the different

samples, a principal component analysis (PCA) was performed(Figure 2C). As expected, biological replicates grouped together,indicating experimental reproducibility. The PCA also revealeddistinct sample populations corresponding to each (sub)com-partment, with the exception of the AS and Emb samples, whichwere partially superimposed (Figure 2C). Interestingly, the twoendosperm interfaces SAL and EAS formed groups that weredistinct both from each other and from the whole endospermsamples. The EASwasmore similar to the whole endosperm than

Maize Embryo/Endosperm Interface Transcriptomics 835

to the SAL, indicating a more similar transcriptomic landscape(Figure 2C).

To explore potential contamination between tissues duringthe dissection process, the expression profiles of previouslyidentified marker genes with tissue-specific expression patternswere investigated (Figures 2D to 2G). Leafy cotyledon1(ZmLec1,Zm00001d017898) andNam/Ataf/Cuc124 (ZmNac124,Zm00001d046126; named ZmNac6 by Zimmermann and Werr[2005]), two embryo-specific genes, were specifically expressedin the embryo samples in our data set (Figure 2D). As expected,ZmLec1 was more strongly expressed in the Emb than in the ASsample (Zhang et al., 2002). Absence of ZmNac124 expression inthe AS was consistent with the strong and specific in situ hy-bridization signal for this gene in the basal part of the embryonicaxis (ZimmermannandWerr, 2005). The twoendosperm-specificgenes ZmZhoupi/Opaque11 (ZmZou/O11, Zm00001d003677)and Opaque2 (O2, Zm00001d018971; Schmidt et al., 1990;Grimault et al., 2015; Feng et al., 2018) were found to be stronglyexpressed in the End and EAS and weakly in the SAL sample(Figure 2E). The weak expression in the Per sample was un-expected but consistent with other transcriptomics data(Sekhon et al., 2011) and could also reflect possible con-tamination of the Per samples with the aleurone layer, sincethe aleurone layer has a tendency to stick to the pericarp (seeDiscussion). In addition, the preferential expression of Al-eurone9 (Al9, Zm00001d012572) and Zm00001d024120genes in the aleurone (Gómez et al., 2009; Li, 2014; Zhanet al., 2015) was reflected by a stronger signal in SALcompared with End (Figure 2F). Al9 and Zm00001d024120also showed a signal in the pericarp samples, again in-dicating a possible contamination of the Per samples by SAL(Figure 2F).

The expression patterns of ESR marker genes (Esr1, Esr2, andEsr3) were also evaluated in our samples. At 13 DAP, the ESRcomprises a small endosperm region situated at the base ofembryo, around the suspensor (Opsahl-Ferstad et al., 1997). Weobserved elevated expression of ESR markers in the SAL and toa lesser extent in the EAS (Figure 2G). Previous in situ hybrid-izations of Esr1 transcripts showed that Esr1 expression is re-stricted to the ESR and absent from the EAS and most if not all ofthe SAL at both 12 and 14 DAP (Opsahl-Ferstad et al., 1997).

However, thebasal part of theSAL is in direct contactwith theESR(Opsahl-Ferstad et al., 1997), and the published data do not ex-clude thepossibility that theEsr1 signalmight extend to theSAL inthis basal part. The apparent elevated expression of ESR markergenes in our SAL transcriptomes may thus reveal contaminationwith adjacent ESRcells during dissection and/or expression in thebasal part of the SAL.In order to compare our full transcriptomic data set with pub-

lished RNA-seq data, we used a unique, spatially resolved maizekernel transcriptome (Zhan et al., 2015). Although different (sub)compartments and developmental stages (8 versus 13DAP) wereused, we retreated both RNA-seq raw data sets using the samebioinformatic pipeline and the same genome version (see Meth-ods) in order to increase comparability. We then performed a PCAon joint data sets. The first principal component (PC1) carries43.7% of the variance and clearly separates the two data sets(Supplemental Figure 3A). It may reflect a batch effect, a combi-nation of the biological effect of the age of sampling (8 versus 13DAP) and technical differences between the two transcriptomes(growing environment, library preparation, etc.). The next com-ponents group together samples from the two data sets and stillcarry a relatively high fraction of the variance (26.9 and 9.7% forPC2 and PC3, respectively). When PC2 was plotted against PC3,13-DAP Emb ismost similar to 8-DAPEmb samples among the 8-DAP samples (Supplemental Figure 3B), indicating that althoughimportant differences exist between these two data sets, thesetwo embryo samples share some similarities in their tran-scriptomic profiles. Likewise, the 13-DAPAS ismost similar to the8-DAP Emb samples among 8-DAP samples (SupplementalFigure3B). The13-DAPSALgroupsmostclosely to the two8-DAPsamples BETL and ESR. Interestingly, the 13-DAP EAS samplesform an independent group that is closer to the two 8-DAP SEsamples (which are the central starchy endosperm [CSE] and theconducting zone [CZ]) among the 8-DAP samples (SupplementalFigure 3B).In summary, we have generated RNA-seq profiles from 13-DAP

maize kernel compartments and embryo/endosperm interfaces.We have made this data available to the community in a user-friendly format via the eFP Browser (http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi?dataSource5Maize_Kernel; seeSupplemental Figure 4 for examples).

Figure 1. Scheme Representing the Six (Sub)compartments Hand-Dissected for Transcriptomics Analysis at Maize Embryo/Endosperm Interfaces.

Ab, abaxial; Ad, adaxial.

836 The Plant Cell

Preferentially Expressed Genes and Biological ProcessesAssociated with Specific Maize Kernel (Sub)compartments

Differential expression analyses were performed between the six(sub)compartments by comparing expression levels between pairsof tissues using a likelihood ratio test with P values adjusted by theBenjamini-Hochbergprocedure tocontrol falsediscovery rates (seeMethods). Genes with both adjusted P values lower than 0.05 andan expression difference of fourfold or greater [log2(fold change)$ 2] were classed as differentially expressed genes (DEGs;

Supplemental Table 2). The full lists of DEGs for the 15 intertissuecomparisons performed are available in Supplemental Data Set 2.To identify the biological processes associated with the DEGs,

a GO analysis was performed. Due to the limited resources

available, a newgenome-wide annotation of all predicted proteins

was performed and linked to GO terms (see Methods). In a first

instance, GO terms enriched in the two zygotic compartments

Emb and End were identified by analyzing DEGs upregulated

in each compartment compared with the two other main

Figure 2. Validation of the RNA-Seq Approach.

(A) and (B)Venndiagrams. For each fraction, the number of genes expressed is indicated: for End, Emb, andPer (A) and for End, EAS, andSAL (B). The totalnumber of genes expressed for all three compartments analyzed are indicated below each Venn diagram.(C) PCA of the 24 RNA samples consisting of four biological replicates of Per, AS, Emb, End, EAS, and SAL.(D) to (G)Graphs represent the expression levels (read counts were normalized using the trimmedmean ofM-value method) in the different samples of thetwo embryo-specific genes ZmLec1 and ZmNac124 (D), the two endosperm-specific genes O2 and ZmZou (E), the two aleurone-specific genes Al9 andZm00001d024120 (F), and the threeEsrgenesEsr1,Esr2, andEsr3 (G).Grayandblackyscalenumbering in (F)are forZm00001d024120andAl9expressionlevels, respectively, and in (G) are for ESr1 and Esr3 (gray) and Esr2 (black).

Maize Embryo/Endosperm Interface Transcriptomics 837

compartments (Table 1). The top 10 GO terms enriched in theDEGs upregulated in the embryo relative to endosperm andpericarp showed a significant enrichment in GO terms related tothe cell cycle, DNA organization, and cytoskeleton organization,consistent with the extensive developmental and mitotic activitywithin the embryo at this stage (Table 1). In contrast, the GO termsenriched in the DEGs upregulated in the endosperm relative toembryo and pericarp were linked to metabolic functions such asnutrient reservoir activity and starch biosynthetic process (Table 1).These enrichments were consistent with the fact that the endo-sperm is a nutrient storage compartment where starch and reserveproteins are synthesized (Nelson and Pan, 1995; Zheng andWang,2015).

Enrichment for Putative Transporters at the Endosperm/Embryo Interface

Focusing in on the embryo/endosperm interfaces, DEGsbetweenthe three subcompartments (AS, SAL, and EAS) and their wholecompartments of origin were identified (Supplemental Table 2). Atotal of 682 genes were found to be differentially expressed be-tween AS and Emb according to the above criteria. Among them,82weremore strongly and600weremoreweakly expressed inAScompared with Emb samples (Supplemental Table 2). As ex-pected, ZmNac124, which is expressed in the coleorhiza (Figures2D, 3C, and 3D; Zimmermann andWerr, 2005), was found amongthe genes showing reduced expression in the apical scutellum.

Only the GO term DNA binding transcription factor activity wasfound tobesignificantly enriched inour analysis in thecomparisonof AS versus Emb (Table 2).The comparison between the EAS and the End revealed 1498

DEGs with 485 genes showing stronger expression in the EASthan in the End and 1013 genes with the inverse profile(Supplemental Table 2). Among the genes more strongly ex-pressed in the EAS, our GO analysis revealed a significant en-richment in only one GO term (GO analysis on molecular functionterms at the F3 level): transmembrane transporter activity (Ta-ble 2), which suggests a stronger expression of transporter-encoding genes in the EAS compared with End.Finally, 2975 genes were found to be differentially expressed

between SAL and End, 1995 corresponding to genes morestrongly expressed in the SAL and 980 corresponding to geneswith lower expression levels in the SAL (Supplemental Table 2).Interestingly, in the first group, our GO analysis revealed an en-richment in two (out of four)GOterms related to transport (Table2).A closer look at gene families encoding transporters among

DEGs confirmed the overrepresentation seen in the GO analysisand revealed differences between the SAL and EAS. Among thegenes that were at least eight times more strongly expressedcompared with End, 8.45% (45/532) of the genes enriched inthe SAL and 16.04% (34/212) of the genes enriched in the EAShave at least one ortholog in rice (Oryza sativa) or in Arabidopsis(Arabidopsis thaliana) that encodes a putative transporter (Ta-ble 3). In the SAL, transcripts of genes encoding MATEs (Multi-antimicrobial extrusion proteins), which have been implicated in

Table 1. Top 10 GO Terms (Sorted by Increasing P Value) Enriched in the DEGs Upregulated in One Main Compartment Compared with the TwoOthers

GO Term Level DEGs/Total Enrichment P

DEGs Emb versus (End and Per): 1,601 of 29,845 genesGO:0010369 chromocenter (C6) (C6) 8/13 11.47 2.11E-09GO:0042555 MCM complex (C3) 9/18 9.32 5.65E-08GO:0003777 microtubule motor activity (F9) 24/144 3.11 1.92E-07GO:0007018 microtubule-based movement (P4) 24/144 3.11 1.92E-07GO:0006928 movement of cell or subcellular component (P3) 24/145 3.09 2.20E-07GO:0098687 chromosomal region (C5) 13/50 4.85 2.34E-07GO:0008092 cytoskeletal protein binding (F4) 42/348 2.25 3.35E-07GO:0003774 motor activity (F8) 24/149 3.00 3.76E-07GO:0031492 nucleosomal DNA binding (F5) 7/16 8.15 5.89E-07GO:0000786 nucleosome (C4) 19/105 3.37 6.85E-07

DEGs End versus (Emb and Per): 818 of 29,845 genesGO:0045735 nutrient reservoir activity (F2) 11/47 8.54 3.59E-09GO:0019252 starch biosynthetic process (P8) 7/27 9.46 4.30E-07GO:0019863 IgE binding (F5) 3/4 27.36 5.60E-07GO:0019865 Ig binding (F4) 3/4 27.36 5.60E-07GO:0004866 endopeptidase inhibitor activity (F6) 9/55 5.97 2.17E-06GO:0010466 negative regulation of peptidase activity (P7) 9/55 5.97 2.17E-06GO:0010951 negative regulation of endopeptidase activity (P8) 9/55 5.97 2.17E-06GO:0030414 peptidase inhibitor activity (F5) 9/55 5.97 2.17E-06GO:0052548 regulation of endopeptidase activity (P7) 9/55 5.97 2.17E-06GO:0061135 endopeptidase regulator activity (F5) 9/55 5.97 2.17E-06

Emb, embryo; End, endosperm; Per, pericarp. Level indicates minimal depth of the GO term in the GO tree, where P5 biological process, F5molecularfunction, and C 5 cellular component. DEGs/Total indicates the number of genes associated with the GO term in the DEGs list/the number of GO term-annotated genes expressed in at least one sample. Enrichment is defined in Methods.

838 The Plant Cell

a diverse array of functions (for review, seeUpadhyay et al., 2019),and ABC (ATP binding cassette) transporters were found tobe the most strongly enriched, whereas in the EAS, genes en-coding transporters from the MtN21/UMAMIT (Usually MultipleAcids Move In And Out Transporter), MtN3/SWEET, and ABCtransporter families were the most represented. When looking atthe putative molecules transported, a large number of genesencoding putative amino acid transporters were found to showstronger expression in the EAS than in the End samples, althoughgenes encoding transporters for various other molecules, in-cluding sugars, heavy metals, phosphate, inorganic ions, or nu-cleotides, also showed stronger expression (Table 3). Regardingthe comparison ofSAL versusEnd, transportersmainly annotatedas involved in amino acid and inorganic ions transport wereidentified (Table3). In summary, ourworkshows thatbothSALandEAS cells strongly express putative transporter-encoding genes,

suggesting that these cells are characterized by an elevatedtransmembrane transport of various molecules and potentiallymediate nutrient repartitioningaround theembryo.However, eachtissue preferentially expresses different classes of transporters,with MtN21/UMAMIT and MtN3/SWEET transporters involved inamino acid and/or sugar transport, respectively, more likely to beenriched in the EAS.

The EAS Is Restricted to One to Three Endosperm CellLayers Adjacent to the Scutellum

The SAL has both cellular and biochemical characteristics of thealeurone, making it inherently different from other endospermtissues (Zheng and Wang, 2014; Gontarek and Becraft, 2017). Incontrast, EAS cells have not been reported to have distinct fea-tures that allow them to be distinguished cytologically from SE

Figure 3. In Situ Hybridization on 13-DAP Maize Kernel Probes.

ProbesdetectingGFP (negative control; [A]and [B]),Zmnac124 (positive control; [C]and [D]),Sweet14a ([E]and [F]),Sweet15a ([G]and [H]),Umamit_eas1([I] and [J]),Pepb11 ([K] and [L]), Zm00001d017285 ([M] and [N]), andScl_eas1 ([O] and [P]) are shown. Arrows indicatemain in situ hybridization signals.emb, embryo; end, endosperm; ped, pedicel; per, pericarp. Bars5500mmin (A), (C), (E), (G), (I), (J), (K), (M), and (O) and1000mm in (B), (D), (F), (H), (L), (N),and (P).

Maize Embryo/Endosperm Interface Transcriptomics 839

cells, which constitute the majority of the volume of the endo-sperm (Van Lammeren, 1987). However, our transcriptomicanalysis suggests that these cells deploy a specific geneticprogram. In order to (1) confirm EAS expression specificity and (2)provide a more precise spatial resolution to define and charac-terize this new region, in situ hybridizations were performed witha set of six genes more than 10-fold enriched in the EAS tran-scriptome compared with the End transcriptome (SupplementalTable 3; see Supplemental Figure 4 for two examples of the eFPbrowser pattern). Three of these genes encode putative trans-porters, namely Sweet14a (Zm00001d007365) and Sweet15a(Zm00001d050577), encoding putative sugar transporters of theSWEET family, and Zm00001d009063, called Umamit_eas1,encoding a putative amino acid transporter belonging to the

UMAMIT family (Müller et al., 2015; Sosso et al., 2015). The threeremaining genes were Phosphatidylethanolamine binding pro-tein11 (Pebp11, Zm00001d037439), a Serine carboxypeptidase-like (Zm00001d014983 or Scl_eas1), and Zm00001d017285,a gene with no name and unknown function (Supplemental Ta-ble 3). The negative control chosen for in situ hybridizations wasan antisense probe generated against a GFP-encoding openreading frame. The positive control was ZmNac124, which isspecifically expressed in the Emb compartment in our tran-scriptome (Figure 2D; Supplemental Table 3) and which hadpreviously been shown by in situ hybridization to be expressed inspecific embryonic tissues (Zimmermann and Werr, 2005). In situhybridizationswereperformedon13-DAPkernels, thesamestageas used for the transcriptome analysis. The four probes detecting

Table 2. All GO Terms from F3 (Molecular Function at Level 3) Significantly Enriched in the DEGs Upregulated in a Subcompartment Compared withits Compartment of Origin

GO Term Level DEGs/Total Enrichment P

DEGs AS versus Emb: 82 of 29,845 genesGO:0003700 DNA binding transcription factor activity (F3) 8/743 3.91 0.000202

DEGs EAS versus End: 485 of 2,9845 genesGO:0022857 transmembrane transporter activity (F3) 26/1,111 1.44 0.0256

DEGs SAL versus End: 1,995 of 29,845 genesGO:0008289 lipid binding (F3) 24/183 1.96 0.000529GO:0003700 DNA binding transcription factor activity (F3) 70/743 1.41 0.00158GO:0022857 transmembrane transporter activity (F3) 97/1,111 1.31 0.00305GO:0005319 lipid transporter activity (F3) 4/30 1.99 0.0468

Emb, embryo; End, endosperm. Level indicates minimal depth of the GO term in the GO tree, where F 5 molecular function. DEGs/Total indicates thenumber of genes associated with the GO term in the DEGs list/the number of GO term-annotated genes expressed in at least one sample. Enrichment isdefined in Methods.

Table 3. Number of Genes Encoding Putative Transporters in the DEGs Upregulated in the SAL or in the EAS Compared with the Endosperm perFamily and per Molecules Putatively Transported

Transporter Family Ratio SAL/End > 8 Ratio EAS/End > 8

MtN21/UMAMIT 1 5MtN3/SWEET 0 3AAP 1 2MATE 7 1ABC 3 4GDU 1 2VIT 0 2Phosphate transporters 0 2Other 32 13Total No. 45 34Percentage in the gene list 8.45% 16.04%Molecules Putatively Transported Ratio SAL/End > 8 Ratio EAS/End > 8Amino acids and/or auxin 7 12Nucleotides 1 1Heavy metal 3 3Sugar 0 4Phosphate 0 2Other inorganic ions 5 2

Analysis was done base on orthology to rice and Arabidopsis (see Methods).

840 The Plant Cell

Sweet15a (Figures 3G and 3H), Pepb11 (Figures 3K and 3L),Zm00001d017285 (Figures 3Mand3N), andScl_eas1 (Figures 3Oand 3P) gave a strong signal restricted to a few layers of endo-sperm cells immediately adjacent to the scutellum, with little or noexpression detected elsewhere in the kernel. For the probe di-rectedagainstSweet14a, the signalwasstrong in theEASbutwasalsopresent, albeitmoreweakly, inother kernel tissues, especiallyin the embryo andaleurone (Figures 3Eand3F). Theprobe againstUmamit_eas1 gave a weaker signal restricted to the apical part ofthe EAS region, consistent with the lower expression levels of thisgene in our transcriptome data (Supplemental Table 3). However,the signal for Umamit_eas1 was specific to these EAS cells(Figures 3I and 3J). These results confirmed that EAS cells havea specific transcriptional program and that this program (and thusthe EAS) is restricted to one to three layers of endosperm cellsadjacent to the scutellum.

The EAS Is a Dynamic Region Reflecting the Period ofStrong Embryo Growth

To evaluate the dynamics of gene expression in the EAS duringkernel development, in situ hybridizations were performed onkernels at different developmental stages (9, 11, 14, 17, and 20DAP; Figure 4; Supplemental Figure 5). The four probes givingstrong and EAS-specific signal at 13 DAP (Sweet15a, Pepb11,Zm00001d017285, and Scl_eas1) were used (Figure 4). In 9-DAPkernels, the probes for Pepb11 and Scl_eas1 showed no signal,whereas those forSweet15aandZm00001d017285gaveastrongsignal in the endosperm cells adjacent to the apical part of theembryo (Figure 4; Supplemental Figure 5). This signal was re-stricted to a few layers of cells in the vicinity of the nascentscutellum. At this stage, the basal part of the embryo was stillsurrounded by ESR cells, and no signal was detected in this re-gion. At 11 DAP, all four probes tested gave a very strong signal inthe layers of endosperm cells adjacent to the scutellum. At 14 and17 DAP, the signal was still detected and restricted to the celllayers in close contact with the embryo (Figure 4; SupplementalFigure 5). Finally, at 20 DAP, the signal decreased for all fourprobes, with a total disappearance for Sweet15a. Together, theseresults revealed that the EAS transcriptomic region was restrictedto adefined timewindow. Its onset at 9DAPwasconcomitantwiththe formation of the scutellum, marking a switch in embryo/en-dosperm interactions from an ESR/embryo to an EAS/scutelluminterface. Its decline occurred around 20DAP,when rapid embryogrowth comes to an end.

EAS Cells Originate from the SE and Undergo Cell Death

Despite the preferential or specific expression of EAS markergenes, and consistent with their SE-like morphology, EAS cellsalso showed some transcriptomic characteristics of the SE, suchas a strong expression of genes encoding ZEIN storage proteins(Supplemental Figure 6). The presence of Zein transcripts in theEAS region is supported by in situ hybridization data (Woo et al.,2001). In order to perform amore global comparison, we asked towhich samples from the Zhan et al. (2015) data set (at 8 DAP) ourEAS transcriptome was most similar, using PCA (Supplemental

Figure 3). Interestingly, onPC3, EASat 13DAPwasmost similar totwo specific SE subregions at 8 DAP: the CSE and the CZ(Supplemental Figure 3B). As EAScells, bothCZandCSEhave nostriking morphological characteristics differentiating them fromthe SE, strengthening the idea that EAS originate from the SE.To address the question of EAS cell fate in proximity to the

scutellum, sagittal sections of the EAS/scutellum interface wereboth hybridized with an EAS-specific probe (against Sweet15atranscripts) and stained with calcofluor to reveal cell walls(Figures 5A and 5B). The accumulation of cell wall materialoccurred at the endosperm interface with the scutellum, whichmay result from the compaction of crushed endosperm cells.Interestingly, in situ hybridization signal for the EAS markergenes was found in the first uncrushed cell layer (Figure 5B). Anappealing model is that EAS cells are actually SE cells that areforced into juxtaposition with the scutellum because of the in-vasive growth of the embryo into the SE during kernel de-velopment (Figure 6), suggesting that the EAS program may notbefixedwithin a static groupof cells but insteadmaybe triggeredas SE cells enter into contact with the scutellum.If this model is correct, EAS cells would be likely to be suc-

cessively eliminated as they come into contact with the em-bryo. Terminal deoxynucleotidyl transferase dUTP nick endlabeling (TUNEL) assays were performed on 15-DAP kernels tovisualize DNA degradation as a potential indicator of thepresence of dying cells. However, it should be noted thatTUNEL signals are neither a fully reliable indicator of all forms ofcell death nor diagnostic of specific cell death programs(Charriaut-Marlangue and Ben-Ari, 1995; Labat-Moleur et al.,1998). In addition to the principal component and coleoptileregions, both of which had previously been shown to givestrong TUNEL signals (Giuliani et al., 2002; Kladnik et al., 2004),we also observed a clear TUNEL-positive signal in some EAScells in close contact with the scutellum (Figures 5C and 5D).This result is consistent with the possibility that a form of celldeath occurs at this interface.To clarify whether transcriptional activation of EAS-specific

genes is linked to the initiation of known cell death pro-grams, the expression levels of genes associated with pro-grammed cell death in plants were analyzed (SupplementalFigure 7; Fagundes et al., 2015; Arora et al., 2017). Surprisingly,orthologs of none of the previously identified programmed celldeath-associated genes were found to be particularly upre-gulated in theEAScomparedwith other samples. In addition, noenrichment of GO terms associated with programmed celldeath was found in the DEGs strongly expressed in the EASrelative to the End samples. Similar results were obtained whencomparing genes strongly expressed in the EAS relative to theEmb, which remains alive (the GO term programmed cell death[GO:0012501] was slightly enriched [ratio of 1.56], but in a notstatistically significant manner [P 5 0.098]). These data sug-gested that either only a small proportion of EAS cells undergocell death or that crushing of EAS cells does not triggera classical programmed cell death program. A parallel could bedrawn with accidental cell death defined in animals, in whichcells die as a result of their immediate structural breakdowndueto physicochemical, physical, or mechanical cues (Galluzziet al., 2015).

Maize Embryo/Endosperm Interface Transcriptomics 841

Impaired Expression of Some EAS Marker Genes inemb Mutants

To test to what extent the proximity of the embryo/scutellum wasrequired for EAS gene expression, the embryo specific mutationemb8522wasused in anR-scm-2genetic backgroundenhancingthe early embryo-deficient phenotype (Sosso et al., 2012). In thisbackground, the recessive emb8522mutation produced vestigialembryoscomposedof asmall heapof cells.Nevertheless, acavity

corresponding to the size of a normal embryo was generated thatwas only very partially occupied by the aborting embryo (Heckelet al., 1999; Sosso et al., 2012). Self-fertilization of heterozygousplants carrying the emb8522mutation was performed, and in situhybridizations were performed on 13-DAP sibling kernels witheither phenotypically wild-type ormutant embryos to visualize thetranscripts of four EAS marker genes (Figure 7). Similar EAS-specific expression patterns were observed in R-scm-2 kernels

Figure 4. In Situ Hybridization of Four Probes Detecting EASMarker Genes (Sweet15a, Pepb11, Zm00001d017285, and Scl_eas1) on Kernel Sections atDifferent Developmental Stages.

Probe detectingGFPwasusedas anegative control. Images are zoomed fromSupplemental Figure 5. For each image, the nameof theprobe is indicated atthe top and the stage on the left. Arrows indicatemain in situ hybridization signals. emb, embryo; end, endosperm; nu, nucellus; ped, pedicel; per, pericarp.Bars 5 200 mm for 9-DAP kernels and 500 mm for the other stages.

842 The Plant Cell

withembryos (Figure7) to thoseobserved inB73kernels (Figures3and 4) for all genes tested, indicating a conservation of EAS cellidentity in this genetic background. In emb kernels, the probesdetectingZm00001d017285andSweet15astill showedasignal inthe EAS region but with an altered distribution (Figure 7). In embkernels, Zm00001d017285 expression was found to be restrictedto the apical part of the embryo cavity and Sweet15a expressionexpanded to the SAL, suggesting an inhibitory role of the normalembryo on Sweet15a expression in the SAL tissue. Interestingly,the two other EAS marker genes tested showed either only veryweak expression (Scl_eas1) or no expression (Pepb11) in embkernels, indicating a promoting effect of the normal embryo on theexpression of these two genes (Figure 7).

DISCUSSION

Transcriptomes at Embryo/Endosperm Interfaces

As in other flowering plants, seed development in maize is gov-erned by specific temporal and spatial genetic programs, dis-tinguishing early development, filling, and maturation on the onehand and embryo, endosperm, and pericarp on the other (Downset al., 2013; Lu et al., 2013; Chen et al., 2014; Li et al., 2014; Quet al., 2016;Meng et al., 2018). Recently, a transcriptome analysisof the nucellus (including the fertilized embryo sac) increased thetemporal resolution and allowed unprecedented access to in-formation regarding thegenetic control of early seeddevelopment(Yi et al., 2019). The most detailed spatial analysis to date usedlaser-capturemicrodissectionon8-DAPkernels (Zhanetal., 2015)to reveal the expression of specific populations of genes in thematernal tissues, the embryo, and themain endosperm cell types,namely ESR, BETL, AL, and SE (which was subdivided into CSEand CZ). Although providing an extremely valuable resource,these studies did not address the question of whether specifictranscriptional domains exist at embryo/endosperm interfaces.The endosperm and embryo are complex compartments with

several morphologically and functionally distinct domains (Olsen,2004; Sabelli and Larkins, 2009). Because they undergo complexand coordinated developmental programs, the interfaces be-tween the embryo and the endosperm represent important, andconstantly changing, zones of exchange, both in term of nutritionand communication (Nowack et al., 2010; Ingram and Gutierrez-Marcos, 2015; Widiez et al., 2017). In order to understand theseinteractions, two subdomains of the endosperm and one sub-domainof theembryowerehand-dissected: theSALat theadaxialside of the embryo; the SE in close contact with the abaxial side oftheembryo (EAS); and the scutellumof theembryo (AS). Kernels at13 DAP were chosen for our analysis because at this stage theembryo has emerged from the ESR and is establishing new in-teractionswith endosperm. Fromapractical point of view, 13DAPis also the earliest stage allowing reliable hand dissection of thechosen interfaces.Contaminationwith neighboring tissues is an important issue in

any dissection experiment. For example, in Arabidopsis, an ex-tremely valuable and globally very reliable resource generated bylaser-capture microdissection (Le et al., 2010; Belmonte et al.,2013) was recently shown to contain some inter-compartmentcontamination, which caused problems for the investigation ofparental contributions to the transcriptomes of early embryos andendosperms (Schon and Nodine, 2017). In our study, precautionswere taken to limit intertissue contamination by (1) washing eachsample before RNA extraction (see Methods) and (2) generatingfour biological replicates for each tissue. Marker gene analysisconfirmed the conformity of the samples, with the exception ofa potential minor contamination of pericarp by the AL, suggestedby the apparent expression of both theAl9 andZm00001d024120aleuronemarker genes and the endospermmarker genesZmZou/O11 andO2 in the pericarp sample (Figures 2E and 2F). This couldhave been caused by the tendency of the AL to stick either to theSE or to the pericarp. In addition, residual ESR tissues at 13DAP might have contaminated both our SAL and EAS samples(Figure 2G).

Figure 5. Crushed Cell Walls and Cell Death Occur in the EAS.

(A) and (B)Calcofluor stainingof cell walls of 13-DAPmaize kernel sections(A) together with in situ hybridization with Sweet15a antisense probes (B)on sagittal sections. Solid white arrows indicate the accumulation ofcrushed cell walls, and empty black arrows indicate in situ hybridizationsignal.(C) and (D) TUNEL labeling of 15-DAP kernels. Fluorescein labeling of theTUNEL-positive nuclei is shown in green and propidium iodide counter-staining in purple. Arrows indicate the nucleus stained by TUNEL in theEAS.emb, embryo; end, endosperm.Bars5200mmin (A)and (B), 500mmin (C),and 100 mm in (D).

Maize Embryo/Endosperm Interface Transcriptomics 843

The EAS, an Endosperm Subdomain Likely Involved inCarbon and Nitrogen Fluxes from the Endosperm tothe Embryo

Transcriptomic profiling of the two endosperm interfaces withthe embryo (SAL and EAS) revealed specific transcriptionalsignatures. While this could have been expected for the cy-tologically distinct SAL, it was rather unexpected for the celllayers adjacent to the abaxial side of the embryo, which do notpresent any obvious cytological differences from other SEcells (Van Lammeren, 1987). Based on the observed enrich-ment of hundreds of transcripts in these cell layers, they rep-resent a novel subdomain of the endosperm, which we namedthe EAS.GO analysis revealed a significant enrichment in the GO cat-

egory transmembrane transporter activity for both the SAL andEAS and additionally for lipid transporter activity for SAL (Ta-ble 2). A closer look at DEGs for both EAS and SAL shows thepresence of different transporter gene families (Table 3). In-terestingly, many Umamits and Sweets, thought to transportamino acids/auxin and sugars, respectively, were found to beenriched in the EAS. UMAMITs and SWEETs are considered tobe bidirectional transporters, although they tend to act as ex-porters when located at the plasma membrane, exporting nu-trients down concentration gradients generated by sinks inadjacent tissues (Chen et al., 2012; Müller et al., 2015). Twononexclusive hypotheses could explain the elevated expressionof transporter-encoding genes in the EAS: either these cellsactively take upnutrients that arrive from theBETL via theSEandthen export them into the apoplastic space surrounding thegrowing embryo, or they are simply involved in recycling nu-trients from dying endosperm cells that are crushed by thegrowing embryo.With regard to nutrient uptake on the embryo side, one might

expect the expression of genes encoding nutrient importers atthe surface of the scutellum in order to take up apoplasticmetabolites.However, in ourAS transcriptome,wewere not ableto detect differentially expressed importer-encoding genes withrespect to the entire Emb. While this could suggest that theregulation of importer activity does not occur at the transcrip-tional level, it seems more likely that our transcriptomic com-parison AS versus Emb was not well designed for theidentification of such genes, since the whole embryo is mainlycomposed of scutellum tissues.In the future, a more detailed comparison of the gene expression

profiles of theBETL (import) and theEAS (export) regions could beinformative. The BETL is an interface specialized in nutrienttransfer from maternal phloem terminals to the endosperm(Chourey andHueros, 2017). The hexose transporter SWEET4c ispreferentially expressed in the BETL, and loss of function ofSweet4c results in the production of a shriveled endosperm,

Figure 6. Scheme Summarizing the EAS Dynamic.

Three different consecutive times points (t0, t1, and t2) are represented.Embryo scutellum invades (representing by arrows) the surrounding SEcells, which enter in cell death (yellow stars). The endosperm cell layers in

contact with the embryo scutellum are regularly eliminated, resulting inan accumulation of crushed cell walls. Additional endosperm cells arethus recruited as EAS, as the embryo grows. Three cells are labeled bya cross pattern to illustrates this dynamic. Emb, embryo scutellum; End,endosperm.

844 The Plant Cell

illustrating the critical importance of hexose transport in theBETL for normal endosperm growth (Sosso et al., 2015). In-terestingly, the Sweet4c gene is also found in the DEGs,showing strong expression in the EAS compared with the en-dosperm as a whole, possibly suggesting commonalities be-tween BETL and EAS function. EAS-specific knockdown ofSweet4c might be one strategy to test this hypothesis and toaddress the question of possible redundancy with Sweet14aand Sweet15a, also enriched in the EAS. Nonetheless, notabledifferences exist between the EAS and the BETL. First, BETLcells have structural features including dramatic cell wall in-growths that make them unique in the endosperm (Leroux et al.,2014; Chourey andHueros, 2017). In contrast, EAS cells cannotbe morphologically differentiated from the SE (Van Lammeren,1987). Second, the BETL represents a static interface, contraryto theEAS,which is displacedas the embryo scutellumexpands(Figure 6) during the most rapid growth phase of the embryo(Chen et al., 2014).

The EAS Is a Developmentally Dynamic Interface

Thedetection ofDNA fragmentation, a characteristic of cell death,in EAS cells (Figures 5C and 5D) together with an accumulation ofcell wall material in this zone (Figures 5A and 5B) suggested thatendosperm cells are eliminated as the embryo grows. An im-portant question is whether this involves a genetically controlledcell-autonomous death or a more atypical and passive cell deathprocess caused by embryo growth. In the Arabidopsis seed,where most of the endosperm degenerates during seed de-velopment, the expression of developmental cell death markergenes such as PLANT ASPARTIC PROTEASE A3 or BI-FUNCTIONAL NUCLEASE1 has been detected at the embryointerface (Olvera-Carrillo et al., 2015; Fourquin et al., 2016). In

maize, less is known about the molecular actors involved in de-velopmental cell death. To the best of our knowledge, cell deathmarker genes have not been comprehensively identified inmaize.Nevertheless, a surveyofputative cell deathmarkergenesderivedfrom comparisons with other plant systems showed their ex-pression in EAS cells, but without any significant enrichmentcompared with other compartments (Supplemental Figure 7;Supplemental Data Set 2). Although cell death in the EAS could betriggered by the activation of unknown cell death-associatedgenes, a more likely explanation for our observations could bea dilution of the transcriptional signal in the EAS transcriptome,making it undetectable. This is supported by TUNEL staining,which revealed a very localized signal limited to a few cells at theimmediate interface with the embryo (Figures 5C and 5D). Inaddition, previous cell death staining with Evans blue did notreveal any massive cell death in the EAS, further supporting thehypothesis of very localized cell death events (Young and Gallie,2000).The precise spatial organization of cell death and transporter

expression remains unclear, but the expression of transportersmight allow the recycling of nutrients from the cells before theydie. As these cells are SE in origin, they could already haveinitiated nutrient storage at 13 DAP, as illustrated by substantialexpression of Zein genes (Supplemental Figure 6). Nutrient re-cycling could be an advantageous way for the plant to efficientlyreuse stored nutrients. Interestingly, in Arabidopsis, the STP13gene, coding forasugar transporter, is upregulated in several celldeath contexts and the expression of many transporters in-crease during organ senescence, suggesting a function in nu-trient recycling from dying cells (Norholm et al., 2006; van derGraaff et al., 2006; Zhang et al., 2014). However, the precise roleof transporters in nutrient recycling remainspoorly understood inplants.

Figure 7. In Situ Hybridization with Several Probes Marking the EAS on 13-DAP Maize Kernel Sections of the R-scm-2 Genetic Background.

ProbedetectingGFPwasusedasanegativecontrol. Kernelscome fromaself-pollinationofamotherplant heterozygous for theemb8522mutation. The toprow (Rscm21emb) corresponds to kernelswith embryo (emb85221/2or1/1), and thebottom row (Rscm2 –emb) corresponds to kernelswithout embryo(emb85222/2). Arrows indicate themain insituhybridizationsignal. emb,embryo; embcav, embryocavitycontaininganabortedembryo; end, endosperm;per, pericarp. Bars 5 1000 mm.

Maize Embryo/Endosperm Interface Transcriptomics 845

The Importance of the Embryo for the Expression of EASMarker Genes

Since the EAS is a mobile interface, forming adjacent to theexpanding scutellum, we asked whether the presence/absenceof the embryo influences the activation of EAS marker genes(Figure 7). In emb8522 mutant kernels, which produce a seem-ingly empty, but normally sized, embryo cavity containing anaborted embryo (Heckel et al., 1999; Sosso et al., 2012), theexpression of different EASmarker geneswasaffected in differentways. TheSweet15agenewasstill expressed inEAScells but alsobecame strongly expressed at the opposite embryo/endosperminterface (SAL). Based on the precedent of the Sweet4c trans-porter gene, which is induced by sugar (Sosso et al., 2015), it ispossible that a similar induction could occur in the case ofSweet15a. The absence of a normal embryo could lead to a build-up of sucrose in the embryo cavity of emb8522mutants, leadingto such an induction. In contrast, the expression domain of theZm00001d017285 marker gene is reduced in emb8522mutants,with expression becoming restricted to the apical part of the EAS.Finally, the expression of Pepb11 and Scl_eas1 is dramaticallyreduced in emb8522mutants comparedwith phenotypically wild-type kernels. Our results suggest that EAS-specific gene ex-pression could be a result of several independent factors, some ofwhich could originate from the endosperm and others from theembryo. The mechanisms involved in embryo cavity formationremain elusive, althougha recent study showed that theSHOHAI1protein is required in the endosperm for the formation of theembryo cavity (Mimura et al., 2018).

Interestingly, the expression of both Pepb11 and Scl_eas1 in-itiates relatively late in the EAS, whereas the expression ofSweet15a and Zm00001d017285 initiates before 9 DAP. Thissuggests the presence of at least two transcriptional programsin the EAS: one initiating early and weakly influenced by theembryo, and a second activated later and more strongly embryo-dependent. The generation of comparable transcriptomes atearlier developmental stages could help us identify the key signalsactivating gene expression in the EAS and potentially pinpointtranscription factors regulating gene expression in this tissue. Inparallel, phenotypic analysis of loss-of-functionmutants of genesenriched in the EAS is needed to further elucidate the biologicalrole of this novel endosperm subdomain.

METHODS

Plant Material and Plant Growth Conditions

The maize (Zea mays) A188 and B73 inbred lines were cultivated in the S2greenhouse with a 16-h illumination period (100 W/m2) at 24/19°C (day/night) and without control of the relative humidity, as described previously(Rousseau et al., 2015; Gilles et al., 2017). The A188 inbred line depicted inSupplemental Figure 1 was cultivated in a growth chamber as describedpreviously (Doll et al., 2019). The emb8522 mutant in the R-scm-2 back-ground (Sosso et al., 2012) and theB73 plants used for in situ hybridizationweregrown inafieldplot locatedat theÉcoleNormaleSupérieure, deLyon,France.

Isolation of Maize Kernel Compartments

Kernel (sub)compartments of theB73 inbred linewere hand-dissected andquickly washed with Dulbecco’s phosphate-buffered saline solution(HyClone, SH30378.02) before freezing them in liquid nitrogen. For each(sub)compartment, four independent biological replicates were produced(Supplemental Table 1). For each biological replicate, the material comesfrom two independent, 13-d-old maize ears [i.e., eight different ears wereused for each (sub)compartment]. Within each biological replicate, tissuesfrom 4 to 84 kernels were pooled depending on the size of the considered(sub)compartment (Supplemental Table 1).

RNA Extraction and RNA-Seq

Total RNAs were extracted with TRIzol reagent, treated with DNase usingthe Qiagen RNase-Free DNase Set, and purified using Qiagen RNeasycolumns according to the supplier’s instructions. RNA-seq librarieswere constructed according to the TruSeq_RNA_SamplePrep_v2_Gui-de_15026495_C protocol (Illumina). Sequencing was performed with anIllumina HiSeq2000 at the Institut de Génomique-Centre National deSéquençage. The RNA-seq samples were sequenced in paired-endmodewithasizingof 260bpanda read lengthof 23100bases.Six sampleswerepooled on each lane of a HiSeq2000 (Illumina) and tagged with individualbar-coded adapters, giving ;62 million pairs per sample. All steps of theexperiment, from growth conditions to bioinformatics analyses, weremanaged in the CATdb database (Gagnot et al., 2008; http://tools.ips2.u-psud.fr/CATdb/) with project identifier NGS2014_21_SeedCom, accord-ing to the minimum information about a high-throughput sequencingexperiment standard (http://fged.org/projects/minseqe/).

RNA-Seq Read Processing and Gene Expression Analysis

RNA-seq reads from all samples were processed using the same pipelinefrom trimming to counts of transcript abundance as follows. Readquality control was performed using the FastQC (S. Andrew, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The rawdata (fastq files)were trimmed using FASTX Toolkit version 0.0.13 (http://hannonlab.cshl.edu/fastx_toolkit/) for Phredquality score >20, read length>30bases, andribosomal sequences were removed with the sortMeRNA tool (Kopylovaet al., 2012).

The genomic mapper TopHat2 (Langmead and Salzberg, 2012) wasused to align read pairs against themaize B73 genome sequence (AGP v4;Jiao et al., 2017) using the gene annotation version 4.32 provided as aGFFfile (Wanget al., 2016). The abundance of each isoformwascalculatedwiththe tool HTSeq-count (Anders et al., 2015) that counts only paired-endreads for which paired-end reads map unambiguously one gene, thusremoving multiple hits (default option union). The genome sequence andannotation file used was retrieved from the Gramene database (http://www.gramene.org/, release 51, in September 2016; Gupta et al., 2016).

Choices for the differential analysis were made based on Rigaill et al.(2018). To increase thedetection power by limiting the number of statisticaltests (Bourgon et al., 2010), we performed an independent filtering bydiscardinggenes that did not haveat least one readafter a count permillionanalysis in at least one-half of the samples. Library size was normalizedusing the method trimmed mean of M-values, and count distribution wasmodeled with a negative binomial generalized linear. Dispersion was es-timated by the edgeRpackage (version 1.12.0;McCarthy et al., 2012) in thestatistical software R (version 2.15.0; R Development Core Team, 2005).Pairwise expression differenceswere performed using likelihood ratio test,and P values were adjusted using the Benjamini-Hochberg procedure tocontrol false discovery rate (Benjamini and Hochberg, 1995). A gene wasdeclared to have a differential expression if its adjusted P value was lowerthan 0.05. The FPKMvalue (fragments per kilobase of transcript permillionmapped reads) is used to estimate and compare gene expressions in eFP

846 The Plant Cell

Browser. This normalization is based on the number of paired-end readsthat mapped each gene, taking into account the gene length and thelibrary size. This RNA-seq read processing method was used for allanalysespresented in thisarticleexcept for thecomparisonofourRNA-seqwith published RNA-seq, for which the data were processed asdescribed below.

Comparison of our RNA-Seq Data Set with a Published RNA-SeqData Set

For the comparison of our data set with previously published RNA-seqdata (Zhan et al., 2015), the raw RNA-seq reads published by Zhan et al.(2015) were retrieved at the National Center for Biotechnology Informa-tion Sequence Read Archive (Leinonen et al., 2011) from BioprojectPRJNA265095 (runs SRR1633457 to SRR1633478). That represents 53million pairs of length 2 3 100 bases for 22 samples. The reads from thetwo data sets were processed using the same pipeline: quality controlwas performed using FastQC version 0.11.7 (S. Andrew, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Sequencing adapterswere clipped using cutadapt v1.16 (Martin, 2011), sequencer artifactswereremoved using FASTX Toolkit version 0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/), andcustomPerl scriptswereapplied to trim regionsof readshaving an average Phred quality score (Ewing andGreen, 1998) lower than28 bases over a sliding window of 4 bases.We noticed that some samplesretrieved from the Sequence Read Archive exhibited a high rRNA content.We built a maize rRNA database by comparing sequences from Silva(Quast et al., 2013) and RFAM (Kalvari et al., 2018) with the maize B73genome sequence; we then used this custom database to filter the RNA-seq reads with sortMeRNA version 2.1b (Kopylova et al., 2012). Readsshorter than 25 bases at the end of this processing, or with no mate, werediscarded.

The genomic mapper HISAT2 v2.2.0 (Kim et al., 2015) was used to alignread pairs against the maize B73 genome sequence (AGP v4; Jiao et al.,2017) using the gene annotation version 4.40 provided as aGFF file (Wanget al., 2016). A first mapping pass was performed with the complete set ofread pairs to discover unannotated splicing sites before the per-samplemapping, with options -k 10-no-discordant -no-softclip and allowing in-trons of length 40 to 150,000 bp. Mapped reads were counted by gene(not distinguishing isoforms) using FeatureCounts (Liao et al., 2014). Thegenome sequence and annotation file used was retrieved from the Gra-menedatabase (http://www.gramene.org/, release51, inSeptember 2016;Gupta et al., 2016).

Normalization, differential analysis, and PCAs were performed withDESeq2 (Love et al., 2014) under R version 3.6.2 (R Development CoreTeam, 2005). The PCAs were done using the 1000 genes with the highestvariance, after applying the variance stabilization transformationdescribedby Anders and Huber (2010) and implemented in DESeq2 v1.24.0. Inparallel, FPKM values and confidence intervals were estimated usingCufflinks version 2.2.1 (Roberts et al., 2011) with options -frag-bias-correct-multi-read-correct-max-multiread-fraction 1.

Venn Diagrams

For each compartment/subcompartment, themean expression of the foursamples was calculated. If the value of the normalized read counts wasequal or superior to one, the gene was considered as expressed. Venndiagrams were drawn using tools available at http://bioinformatics.psb.ugent.be/webtools/Venn/.

Functional Annotation of the Maize Transcriptome and GO TermEnrichment Analysis

The maize B73 genome sequence v4 (Jiao et al., 2017) and the geneannotation v4.40 were used to predict transcript sequences using thegffread script from the Cufflinks package v2.2.1 (Trapnell et al., 2013). Ineach isoform sequence, the putative open reading frames were identifiedusing TransDecoder (Haas et al., 2013; https://github.com/TransDecoder/TransDecoder/wiki), and the amino acid sequence was predicted. From46,272 genes, 138,270 transcripts were predicted, leading to 149,699amino acid sequences.

The predicted protein sequences were annotated for functionaldomainswith InterProScanv5.27-66.0 (Joneset al., 2014) usingdatabasesPfamv31.0 (Punta et al., 2012) andPanther 12.0 (Mi et al., 2013). Theywerealso comparedwithUniProtKBprotein database version 2017_12 (UniProtConsortium, 2019). The completeSwiss-Prot databaseof curated proteinswas used, (containing 41,689 plant sequences and 514,699 nonplantsequences) but only the plant subset of the noncurated database TrEMBL(containing 5,979,810 sequences). The comparison was performed usingWU-BlastP v2.0MP (Altschul et al., 1990) with parametersW53 Q57 R52matrix5BLOSUM80 B5200 V5200 E51e-6 hitdist560 hspsepqmax530hspsepsmax530 sump postsw. BLAST output was filtered using customPerl scripts to keep only matches with log10(e-value) no lower than 75% ofthe best log10(e-value). GO terms (Ashburner et al., 2000) associated withmatched proteins were retrieved from AmiGO (Carbon et al., 2009) with alltheir ancestors in the GO graph, using the SQL interface. For each maizeprotein, we kept theGO terms associatedwith all itsmatchedproteins or atleast with five matched proteins. For each maize gene, we merged the GOterms of all its isoforms.

For subsets of genes selected based on their expression pattern, weusedourGOannotation toperformanenrichmentanalysis. Theenrichmentof a gene subset in a specific GO term is defined as follows:

R5

ðGenes annotated with the GO term in the subsetÞ=ðTotal genes in the subsetÞ

ðGenes annotated with the GO term among all expressed genesÞ=ðTotal expressed genesÞ

A hypergeometric test (R version 3.2.3; R Development Core Team, 2005)was applied to assess the significance of enrichment/depletion of eachsubset (Pavlidis et al., 2004; Falcon and Gentleman, 2007). Custom Perlscripts using GraphViz (Ellson et al., 2001; https://graphviz.gitlab.io/) wereused to browse the GO graph and identify enrichments or depletions thatwere both statistically significant and biologically relevant. Only geneswithat least one match on UniProt and only GO terms with at least one gene inthe subset were considered for all those statistical tests.

Analysis of Gene Categories and Orthology

Analysis of orthology to rice (Oryza sativa) and Arabidopsis (Arabidopsisthaliana; Table 3) was based on Maize GDB annotations (https://www.maizegdb.org/; Andorf et al., 2016). The Zein genes were selectedbased on a previous gene list (Chen et al., 2014, 2017) and on Gramenedatabase annotations (http://www.gramene.org/; Gupta et al., 2016). Thelist of cell death-associated geneswas based on previously published lists(Fagundes et al., 2015; Arora et al., 2017). Heat maps were drawn with theonline Heatmapper tool (http://www2.heatmapper.ca/; Babicki et al.,2016).

Kernel Fixation and in Situ Hybridization

Kernels were fixed in 4% (w/v) paraformaldehyde (pH 7 adjusted with H2

SO4) for 2 h under vacuum. For increased fixation efficiency, the two uppercorners of the kernels were cut and vacuum was broken every 15 min.Kernels were dehydrated and included with Paraplast according to the

Maize Embryo/Endosperm Interface Transcriptomics 847

protocol described by Jackson (1991). Sections of 10 to 15 mm were cutwith an HM355S microtome and attached on Adhesion Slides SuperfrostUltra plus (ThermoFisher Scientific). RNA probes were amplified fromgenomic DNA or cDNA (Supplemental Table 4) and labeled by digoxigenin(DIG) using the T7 reverse transcriptase kit of Promega, according tocompany instructions. RNA probes were then hydrolyzed in carbonatebuffer (120 mM Na2CO3 and 80 mM NaHCO3) at 60°C for various timesdepending on the probe length (Supplemental Table 4) in order to obtainRNA fragments between 200 and 300 nucleotides (Jackson, 1991).

For the prehybridization of the sections, the protocol described byJackson (1991) was followed with some slight changes: pronase wasreplaced by proteinase K (1 mg/mL; ThermoFisher Scientific) in its buffer(100 mM Tris and 50mMEDTA, pH 8), and formaldehyde was replaced byparaformaldehyde as described above. For each slide, 1 mL of RNA probewas diluted in 74mL of DIG EasyHyb buffer (Roche), denatured for 3min at80°C, and dropped on a section that was immediately covered by a coverslip. Hybridization was performed overnight at 50°C in a hermeticallyclosed box. Initial posthybridization treatments were performed usinggentle shaking as follows: 0.13 SSC buffer (from stock solution 203 SSC[3 M NaCl and 300 mM trisodium citrate, adjusted to pH 7 with HCl]) and0.5%(v/v)SDS for30minat50°C to remove thecover slips.Twobathswereused of 1.5 h in 23 SSC buffer mixed with 50% formamide at 50°C andfollowed by 5 min in Tris-buffered saline (TBS) buffer (400 mM NaCl and0.1mMTris-HCl, pH 7.5) at room temperature. Slides were then incubatedin0.5%(w/v)blocking reagentsolution (Roche) for 1h, followedby30min inTBS buffer with 1% (w/v) BSA and 0.3% (v/v) Triton X-100. Probe im-munodetection was performed in a wet chamber with 500 mL per slide of0.225 units/mL anti-DIG antibodies (Anti-Digoxigenin-AP, Fab fragments;Sigma-Aldrich) diluted in TBS with 1% (w/v) BSA and 0.3% (v/v) Triton X-100. After 1.5 h of incubation, slides were washed three times for 20min inTBS buffer with 1% (w/v) BSA and 0.3% (v/v) Triton X-100 and equilibratedin buffer 5 (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, and 50 mM MgCl2).Revelation was performed overnight in darkness in a buffer with 0.5 g/Lnitroblue tetrazolium and 0.2 g/L 5-bromo-4-chloro-3-indolyl phosphate.Slideswere finally washed four times inwater to stop the reaction andwereoptionally stained with calcofluor (fluorescent brightener 28; Sigma-Aldrich)and mounted in entellan (VWR). Photographs were taken either witha VHX900F digital microscope (Keyence) or for magnification with anAxioImager 2 microscope (Zeiss).

Terminal Deoxynucleotidyl Transferase dUTP Nick EndLabeling Assay

Kernels at 15 DAP were fixed in paraformaldehyde, included in Paraplast,and sectioned as described above. Paraplast was removed by successivebaths in xylene (23 5min), and samples were then rehydrated through thefollowing ethanol series: ethanol 100% (5 min), ethanol 95% (v/v; 3 min),ethanol 70% (v/v; 3 min), ethanol 50% (v/v; 3 min), NaCl 0.85% (w/v) inwater (5 min), and Dulbecco’s PBS solution (5 min). Sections were thenpermeabilized using proteinase K (1 mg/mL; ThermoFisher Scientific) for10minat37°Candfixedagain inparaformaldehyde.Sectionswerewashedin PBS, and terminal deoxynucleotidyl transferase dUTP nick end labelingwas performed with the ApoAlert DNA Fragmentation Assay Kit (Takara)according to the manufacturer’s instructions. Sections were then coun-terstained with propidium iodide (1 mg/mL in PBS) for 15 min in darknessbefore beingwashed three times for 5min inwater. Slidesweremounted inAnti-fade Vectashield (Vector Laboratories). The fluorescein-dUTP in-corporated at the free 39 hydroxyl ends of fragmented DNA was excited at520 nmand propidium iodide at 620 nm. Imageswere taken on a spinning-diskmicroscope, with aCSU22 confocal head (Yokogawa) and an Ixon897EMCCD camera (Andor), on a DMI4000 microscope (Leica).

Accession Numbers

RNA-seq raw data were deposited in the international repository GeneExpressionOmnibus (Edgar et al., 2002; http://www.ncbi.nlm.nih.gov/geo)under project identifier GSE110060. RNA-seq data as FPKM values areavailable via the eFP Browser engine (http://bar.utoronto.ca/efp_maize/cgi-bin/efpWeb.cgi?dataSource5Maize_Kernel), which paints the ex-pression data onto images representing the samples used to generate theRNA-seq data. Custom codes and scripts are available at http://flower.ens-lyon.fr/maize/seedcom/.

Supplemental Data

Supplemental Figure 1. Illustration of hand-dissected maize kernelcompartments and sub-compartments.

Supplemental Figure 2. Proportion of mapped reads andexpressed genes.

Supplemental Figure 3. Relationships between transcriptomic data-sets at 13 DAP (this study), and at 8 DAP (Zhan et al., 2015) assessedby PCA analysis.

Supplemental Figure 4. Example of eFP Browser views

Supplemental Figure 5. Whole kernel views of the in situ hybrid-izations presented in Figure 4.

Supplemental Figure 6. Heat map of Zein precursor gene expression.

Supplemental Figure 7. Heat maps for genes potentially involved inprogrammed cell death.

Supplemental Table 1. Number of kernels used for each of the fourbiological replicates.

Supplemental Table 2. Number of genes differentially expressedbetween a sub compartment and its compartment of origin.

Supplemental Table 3. Mean expression values and gene IDs ofgenes selected for in situ hybridization.

Supplemental Table 4. Primers used in this study and conditions forRNA probes synthesis.

Supplemental Data Set 1. Number of normalized read counts pergene annotated in the AGP v4 version of the B73 maize genome.

Supplemental Data Set 2. Pairwise comparison of gene expressionlevels between the tissues.

ACKNOWLEDGMENTS

We thank Justin Berger, Patrice Bolland, and Alexis Lacroix for maizeculture, Isabelle Desbouchages and Hervé Leyral for buffer and mediapreparation, as well as Jérôme Laplaige, Marie-France Gérentes, andGhislaine Gendrot for technical assistance during sample dissections.We also thank Sophy Chamot and Frédérique Rozier for sharing protocolsfor in situ hybridization. The sequencing platform (POPS-IPS2) benefitsfrom the support of theAgenceNationale de laRecherche (ANR-10-LABX-0040-SPS). We thank the PLATIM imaging facility of the SFR BiosciencesGerland-Lyon Sud (UMS344/US8) and especially Claire Lionnet for herhelp in imagining. We acknowledge support from the Pôle Scientifique deModélisation Numérique of the École Normale Supérieure de Lyon forcomputing resources. This work was supported by the Plant Science andBreeding Division of the Institut National de la Recherche en Agriculture etAlimentation et Environnement (BAP, INRAE, Project SeedCom to T.W.).N.M.D. was supported by a Ph.D. fellowship from the Ministère de

848 The Plant Cell

l’Enseignement Supérieur et de la Recherche. Part of this work has beenrefused once for funding by the Agence Nationale de la Recherche.

AUTHOR CONTRIBUTIONS

N.M.D. and T.W. conceived and designed the experiments; T.W. per-formed sample dissections (Supplemental Figure 1) and RNA extractions;J.C. performed RNA-seq library preparation and sequencing; V.B. per-formedRNA-seq readprocessinganddifferential geneexpressionanalysis(Figure 1C; Supplemental Data Sets 1 and 2; Supplemental Figure 2); J.J.performed bioinformatics to create the GO database and provided scriptsto analyze the GO as well as realized the comparison between publishedtranscriptomes (Supplemental Figure 3); A.G. and N.D.-F. performed theTUNEL assay (Figures 5C and 5D); N.M.D. performed all other remainingexperiments; E.E., A.P., and N.J.P. contributed to the RNA-seq dataaccessibility via the eFP Browser engine; N.M.D., P.M.R., and T.W. ana-lyzed the data; N.M.D. prepared tables and figures; N.M.D., G.C.I., P.M.R.,and T.W. wrote the manuscript; T.W. was involved in project managementand obtained funding.

Received October 1, 2019; revised February 3, 2020; accepted February20, 2020; published February 21, 2020.

REFERENCES

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J.(1990). Basic local alignment search tool. J. Mol. Biol. 215:403–410.

Anders, S., and Huber, W. (2010). Differential expression analysis forsequence count data. Genome Biol. 11: R106.

Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq: A Pythonframework to work with high-throughput sequencing data. Bio-informatics 31: 166–169.

Andorf, C.M., et al. (2016). MaizeGDB update: New tools, data andinterface for the maize model organism database. Nucleic AcidsRes. 44: D1195–D1201.

Arora, K., Panda, K.K., Mittal, S., Mallikarjuna, M.G., Rao, A.R.,Dash, P.K., and Thirunavukkarasu, N. (2017). RNAseq revealedthe important gene pathways controlling adaptive mechanismsunder waterlogged stress in maize. Sci. Rep. 7: 10950.

Ashburner, M., et al. (2000). Gene Ontology: Tool for the unification ofbiology. Nat. Genet. 25: 25–29.

Babicki, S., Arndt, D., Marcu, A., Liang, Y., Grant, J.R.,Maciejewski, A., and Wishart, D.S. (2016). Heatmapper: Web-enabled heat mapping for all. Nucleic Acids Res. 44: W147–W153.

Belmonte, M.F., et al. (2013). Comprehensive developmental profilesof gene activity in regions and subregions of the Arabidopsis seed.Proc. Natl. Acad. Sci. USA 110: E435–E444.

Benjamini, Y., and Hochberg, Y. (1995). Controlling the false dis-covery rate: A practical and powerful approach to multiple testing.J. R. Stat. Soc. Ser. B Methodol. 57: 289–300.

Berger, F. (1999). Endosperm development. Curr. Opin. Plant Biol. 2:28–32.

Berger, F. (2003). Endosperm: The crossroad of seed development.Curr. Opin. Plant Biol. 6: 42–50.

Bezrutczyk, M., Hartwig, T., Horschman, M., Char, S.N., Yang, J.,Yang, B., Frommer, W.B., and Sosso, D. (2018). Impaired phloemloading in zmsweet13a,b,c sucrose transporter triple knock-outmutants in Zea mays. New Phytol. 218: 594–603.

Bommert, P., and Werr, W. (2001). Gene expression patterns in themaize caryopsis: Clues to decisions in embryo and endospermdevelopment. Gene 271: 131–142.

Bourgon, R., Gentleman, R., and Huber, W. (2010). Independentfiltering increases detection power for high-throughput experi-ments. Proc. Natl. Acad. Sci. USA 107: 9546–9551.

Cai, G., Faleri, C., Del Casino, C., Hueros, G., Thompson, R.D., andCresti, M. (2002). Subcellular localisation of BETL-1, -2 and -4 inZea mays L. endosperm. Sex. Plant Reprod. 15: 85–98.

Carbon, S., Ireland, A., Mungall, C.J., Shu, S., Marshall, B., andLewis, S. (2009). AmiGO: Online access to ontology and annotationdata. Bioinformatics 25: 288–289.

Charriaut-Marlangue, C., and Ben-Ari, Y. (1995). A cautionary note on theuse of the TUNEL stain to determine apoptosis. Neuroreport 7: 61–64.

Chen, J., Zeng, B., Zhang, M., Xie, S., Wang, G., Hauck, A., and Lai,J. (2014). Dynamic transcriptome landscape of maize embryo andendosperm development. Plant Physiol. 166: 252–264.

Chen, L.-Q., Qu, X.-Q., Hou, B.-H., Sosso, D., Osorio, S., Fernie, A.R.,and Frommer, W.B. (2012). Sucrose efflux mediated by SWEET pro-teins as a key step for phloem transport. Science 335: 207–211.

Chen, X., Feng, F., Qi, W., Xu, L., Yao, D., Wang, Q., and Song, R.(2017). Dek35 encodes a PPR protein that affects cis-splicing ofmitochondrial nad4 intron 1 and seed development in maize. Mol.Plant 10: 427–441.

Cheng, W.H., Taliercio, E.W., and Chourey, P.S. (1996). The Minia-ture1 seed locus of maize encodes a cell wall invertase required fornormal development of endosperm and maternal cells in the pedi-cel. Plant Cell 8: 971–983.

Chourey, P.S., and Hueros, G. (2017). The basal endosperm transferlayer (BETL): Gateway to the maize kernel. In Maize Kernel De-velopment, B. Larkins, ed (Wallingford, UK: CABI), pp. 56–67.

Davis, R., Smith, J., and Cobb, B. (1990). A light and electron-microscope investigation of the transfer cell region of maize cary-opses. Can. J. Bot. 68: 471–479.

Diboll, A.G., and Larson, D.A. (1966). An electron microscopic studyof the mature megagametophyte in Zea mays. Am. J. Bot. 53:391–402.

Doll, N.M., Depège-Fargeix, N., Rogowsky, P.M., and Widiez, T. (2017).Signaling in early maize kernel development. Mol. Plant 10: 375–388.

Doll, N.M., Gilles, L.M., Gérentes, M.-F., Richard, C., Just, J.,Fierlej, Y., Borrelli, V.M.G., Gendrot, G., Ingram, G.C.,Rogowsky, P.M., and Widiez, T. (2019). Single and multiple geneknockouts by CRISPR-Cas9 in maize. Plant Cell Rep. 38: 487–501.

Downs, G.S., Bi, Y.-M., Colasanti, J., Wu, W., Chen, X., Zhu, T.,Rothstein, S.J., and Lukens, L.N. (2013). A developmental tran-scriptional network for maize defines coexpression modules. PlantPhysiol. 161: 1830–1843.

Dumas, C., and Rogowsky, P. (2008). Fertilization and early seedformation. C. R. Biol. 331: 715–725.

Edgar, R., Domrachev, M., and Lash, A.E. (2002). Gene ExpressionOmnibus: NCBI gene expression and hybridization array data re-pository. Nucleic Acids Res. 30: 207–210.

Ellson, J., Gansner, E., Koutsofios, L., North, S.C., and Woodhull,G. (2001). Graphviz: Open source graph drawing tools. In GraphDrawing: GD 2001, P. Mutzel, M. Jünger, and and S. Leipert, eds(Berlin: Springer-Verlag), pp. 483–484.

Ewing, B., and Green, P. (1998). Base-calling of automated se-quencer traces using phred. II. Error probabilities. Genome Res. 8:186–194.

Fagundes, D., Bohn, B., Cabreira, C., Leipelt, F., Dias, N.,Bodanese-Zanettini, M.H., and Cagliari, A. (2015). Caspases inplants: Metacaspase gene family in plant stress responses. Funct.Integr. Genomics 15: 639–649.

Maize Embryo/Endosperm Interface Transcriptomics 849

Falcon, S., and Gentleman, R. (2007). Using GOstats to test genelists for GO term association. Bioinformatics 23: 257–258.

Feng, F., Qi, W., Lv, Y., Yan, S., Xu, L., Yang, W., Yuan, Y., Chen, Y.,Zhao, H., and Song, R. (2018). OPAQUE11 is a central hub of theregulatory network for maize endosperm development and nutrientmetabolism. Plant Cell 30: 375–396.

Fourquin, C., Beauzamy, L., Chamot, S., Creff, A., Goodrich, J.,Boudaoud, A., and Ingram, G. (2016). Mechanical stress mediatedby both endosperm softening and embryo growth underlies endo-sperm elimination in Arabidopsis seeds. Development 143:3300–3305.

Gagnot, S., Tamby, J.-P., Martin-Magniette, M.-L., Bitton, F.,Taconnat, L., Balzergue, S., Aubourg, S., Renou, J.-P.,Lecharny, A., and Brunaud, V. (2008). CATdb: A public access toArabidopsis transcriptome data from the URGV-CATMA platform.Nucleic Acids Res. 36: D986–D990.

Galluzzi, L., et al. (2015). Essential versus accessory aspects of celldeath: Recommendations of the NCCD 2015. Cell Death Differ. 22:58–73.

Gilles, L.M., et al. (2017). Loss of pollen-specific phospholipase NOTLIKE DAD triggers gynogenesis in maize. EMBO J. 36: 707–717.

Giuliani, C., Consonni, G., Gavazzi, G., Colombo, M., and Dolfini, S.(2002). Programmed cell death during embryogenesis in maize.Ann. Bot. 90: 287–292.

Gómez, E., Royo, J., Guo, Y., Thompson, R., and Hueros, G. (2002).Establishment of cereal endosperm expression domains: Identifi-cation and properties of a maize transfer cell-specific transcriptionfactor, ZmMRP-1. Plant Cell 14: 599–610.

Gómez, E., Royo, J., Muñiz, L.M., Sellam, O., Paul, W., Gerentes,D., Barrero, C., López, M., Perez, P., and Hueros, G. (2009).The maize transcription factor myb-related protein-1 is a keyregulator of the differentiation of transfer cells. Plant Cell 21:2022–2035.

Gontarek, B.C., and Becraft, P.W. (2017). Aleurone. In Maize KernelDevelopment, B. Larkins, ed (Wallingford, UK: CABI), pp. 68–80.

Grimault, A., Gendrot, G., Chamot, S., Widiez, T., Rabillé, H.,Gérentes, M.-F., Creff, A., Thévenin, J., Dubreucq, B., Ingram,G.C., Rogowsky, P.M., and Depège-Fargeix, N. (2015). ZmZHOUPI,an endosperm-specific basic helix-loop-helix transcription factor in-volved in maize seed development. Plant J. 84: 574–586.

Gupta, P., et al. (2016). Gramene database: Navigating plant com-parative genomics resources. Curr. Plant Biol. 7–8: 10–15.

Gutiérrez-Marcos, J.F., Costa, L.M., Biderre-Petit, C., Khbaya, B.,O’Sullivan, D.M., Wormald, M., Perez, P., and Dickinson, H.G.(2004). Maternally expressed gene1 is a novel maize endospermtransfer cell-specific gene with a maternal parent-of-origin patternof expression. Plant Cell 16: 1288–1301.

Haas, B.J., et al. (2013). De novo transcript sequence reconstructionfrom RNA-seq using the Trinity platform for reference generationand analysis. Nat. Protoc. 8: 1494–1512.

Heckel, T., Werner, K., Sheridan, W.F., Dumas, C., and Rogowsky,P.M. (1999). Novel phenotypes and developmental arrest in earlyembryo specific mutants of maize. Planta 210: 1–8.

Hueros, G., Gomez, E., Cheikh, N., Edwards, J., Weldon, M.,Salamini, F., and Thompson, R.D. (1999a). Identification of a pro-moter sequence from the BETL1 gene cluster able to confertransfer-cell-specific expression in transgenic maize. Plant Physiol.121: 1143–1152.

Hueros, G., Royo, J., Maitz, M., Salamini, F., and Thompson, R.D.(1999b). Evidence for factors regulating transfer cell-specific ex-pression in maize endosperm. Plant Mol. Biol. 41: 403–414.

Ingram, G., and Gutierrez-Marcos, J. (2015). Peptide signallingduring angiosperm seed development. J. Exp. Bot. 66: 5151–5159.

Ingram, G.C., Boisnard-Lorig, C., Dumas, C., and Rogowsky, P.M.(2000). Expression patterns of genes encoding HD-ZipIV homeodomain proteins define specific domains in maize embryos andmeristems. Plant J. 22: 401–414.

Jackson, D. (1991). In-situ hybridization in plants. In Molecular PlantPathology: A Practical Approach, D.J. Bowles, ed (Oxford, UK:Oxford University Press), pp. 163–174.

Jestin, L., Ravel, C., Auroy, S., Laubin, B., Perretant, M.-R., Pont,C., and Charmet, G. (2008). Inheritance of the number and thick-ness of cell layers in barley aleurone tissue (Hordeum vulgare L.): Anapproach using F2-F3 progeny. Theor. Appl. Genet. 116: 991–1002.

Jiao, Y., et al. (2017). Improved maize reference genome with single-molecule technologies. Nature 546: 524–527.

Jones, P., et al. (2014). InterProScan 5: Genome-scale protein func-tion classification. Bioinformatics 30: 1236–1240.

Kalvari, I., Argasinska, J., Quinones-Olvera, N., Nawrocki, E.P.,Rivas, E., Eddy, S.R., Bateman, A., Finn, R.D., and Petrov, A.I.(2018). Rfam 13.0: Shifting to a genome-centric resource for-non-coding RNA families. Nucleic Acids Res. 46: D335–D342.

Kang, B.-H., Xiong, Y., Williams, D.S., Pozueta-Romero, D., andChourey, P.S. (2009). Miniature1-encoded cell wall invertase isessential for assembly and function of wall-in-growth in the maizeendosperm transfer cell. Plant Physiol. 151: 1366–1376.

Kiesselbach, T.A., and Walker, E.R. (1952). Structure of certainspecialized tissues in the kernel of corn. Am. J. Bot. 39: 561–569.

Kim, D., Langmead, B., and Salzberg, S.L. (2015). HISAT: A fastspliced aligner with low memory requirements. Nat. Methods 12:357–360.

Kladnik, A., Chamusco, K., Dermastia, M., and Chourey, P. (2004).Evidence of programmed cell death in post-phloem transport cellsof the maternal pedicel tissue in developing caryopsis of maize.Plant Physiol. 136: 3572–3581.

Kopylova, E., Noé, L., and Touzet, H. (2012). SortMeRNA: Fast andaccurate filtering of ribosomal RNAs in metatranscriptomic data.Bioinformatics 28: 3211–3217.

Labat-Moleur, F., Guillermet, C., Lorimier, P., Robert, C.,Lantuejoul, S., Brambilla, E., and Negoescu, A. (1998). TUNELapoptotic cell detection in tissue sections: Critical evaluation andimprovement. J. Histochem. Cytochem. 46: 327–334.

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read align-ment with Bowtie 2. Nat. Methods 9: 357–359.

Le, B.H., et al. (2010). Global analysis of gene activity duringArabidopsis seed development and identification of seed-specific transcription factors. Proc. Natl. Acad. Sci. USA 107:8063–8070.

Leinonen, R., Sugawara, H., and Shumway, M. (2011). The se-quence read archive. Nucleic Acids Res. 39: D19–D21.

Leroux, B.M., Goodyke, A.J., Schumacher, K.I., Abbott, C.P.,Clore, A.M., Yadegari, R., Larkins, B.A., and Dannenhoffer,J.M. (2014). Maize early endosperm growth and development:From fertilization through cell type differentiation. Am. J. Bot. 101:1259–1274.

Li, G., et al. (2014). Temporal patterns of gene expression in de-veloping maize endosperm identified through transcriptome se-quencing. Proc. Natl. Acad. Sci. USA 111: 7582–7587.

Liao, Y., Smyth, G.K., and Shi, W. (2014). featureCounts: An efficientgeneral purpose program for assigning sequence reads to genomicfeatures. Bioinformatics 30: 923–930.

Lopes, M.A., and Larkins, B.A. (1993). Endosperm origin, de-velopment, and function. Plant Cell 5: 1383–1399.

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimationof fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 15: 550.

850 The Plant Cell

Lowe, J., and Nelson, O.E. (1946). Miniature seed: A study in thedevelopment of a defective caryopsis in maize. Genetics 31:525–533.

Lu, X., Chen, D., Shu, D., Zhang, Z., Wang, W., Klukas, C., Chen,L.L., Fan, Y., Chen, M., and Zhang, C. (2013). The differentialtranscription network between embryo and endosperm in the earlydeveloping maize seed. Plant Physiol. 162: 440–455.

Martin, M. (2011). Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.j. 17: 10–12.

McCarthy, D.J., Chen, Y., and Smyth, G.K. (2012). Differential ex-pression analysis of multifactor RNA-seq experiments with respectto biological variation. Nucleic Acids Res. 40: 4288–4297.

Meng, D., Zhao, J., Zhao, C., Luo, H., Xie, M., Liu, R., Lai, J., Zhang,X., and Jin, W. (2018). Sequential gene activation and gene im-printing during early embryo development in maize. Plant J. 93:445–459.

Mi, H., Muruganujan, A., and Thomas, P.D. (2013). PANTHER in2013: Modeling the evolution of gene function, and other gene at-tributes, in the context of phylogenetic trees. Nucleic Acids Res. 41:D377–D386.

Miller, M.E., and Chourey, P.S. (1992). The maize invertase-deficientminiature-1 seed mutation is associated with aberrant pedicel andendosperm development. Plant Cell 4: 297–305.

Mimura, M., Kudo, T., Wu, S., McCarty, D.R., and Suzuki, M. (2018).Autonomous and nonautonomous functions of the maize Shohai1gene, encoding a RWP-RK putative transcription factor, in regula-tion of embryo and endosperm development. Plant J. 95: 892–908.

Müller, B., et al. (2015). Amino acid export in developing Arabidopsisseeds depends on UmamiT facilitators. Curr. Biol. 25: 3126–3131.

Nelson, O., and Pan, D. (1995). Starch synthesis in maize endo-sperms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46: 475–496.

Norholm, M.H.H., Nour-Eldin, H.H., Brodersen, P., Mundy, J., andHalkier, B.A. (2006). Expression of the Arabidopsis high-affinityhexose transporter STP13 correlates with programmed cell death.FEBS Lett. 580: 2381–2387.

Nowack, M.K., Ungru, A., Bjerkan, K.N., Grini, P.E., and Schnittger,A. (2010). Reproductive cross-talk: Seed development in floweringplants. Biochem. Soc. Trans. 38: 604–612.

Olsen, O.-A. (2001). Endosperm development: Cellularization and cellfate specification. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52:233–267.

Olsen, O.-A. (2004). Nuclear endosperm development in cereals andArabidopsis thaliana. Plant Cell 16 (Suppl.): S214–S227.

Olvera-Carrillo, Y., et al. (2015). A conserved core of programmedcell death indicator genes discriminates developmentally and en-vironmentally induced programmed cell death in plants. PlantPhysiol. 169: 2684–2699.

Opsahl-Ferstad, H.G., Le Deunff, E., Dumas, C., and Rogowsky,P.M. (1997). ZmEsr, a novel endosperm-specific gene expressed ina restricted region around the maize embryo. Plant J. 12: 235–246.

Pavlidis, P., Qin, J., Arango, V., Mann, J.J., and Sibille, E. (2004).Using the gene ontology for microarray data mining: A comparisonof methods and application to age effects in human prefrontalcortex. Neurochem. Res. 29: 1213–1222.

Porter, G.A., Knievel, D.P., and Shannon, J.C. (1987). Assimilateunloading from maize (Zea mays L.) pedicel tissues. II. Effects ofchemical agents on sugar, amino acid, and C-assimilate unloading.Plant Physiol. 85: 558–565.

Punta, M., et al. (2012). The Pfam protein families database. NucleicAcids Res. 40: D290–D301.

Qu, J., Ma, C., Feng, J., Xu, S., Wang, L., Li, F., Li, Y., Zhang, R.,Zhang, X., Xue, J., and Guo, D. (2016). Transcriptome dynamicsduring maize endosperm development. PLoS One 11: e0163814.

Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza,P., Peplies, J., and Glöckner, F.O. (2013). The SILVA ribosomalRNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 41: D590–D596.

R Development Core Team (2005). R: A language and environmentfor statistical computing, reference index version 2.2.1. https://www.r-project.org/.

Randolph, L.F. (1936). Developmental Morphology of the Caryopsis inMaize.. (Washington, DC: U.S. Department of Agriculture).

Rigaill, G., et al. (2018). Synthetic data sets for the identification ofkey ingredients for RNA-seq differential analysis. Brief. Bioinform.19: 65–76.

Roberts, A., Trapnell, C., Donaghey, J., Rinn, J.L., and Pachter, L.(2011). Improving RNA-seq expression estimates by correcting forfragment bias. Genome Biol. 12: R22.

Rousseau, D., Widiez, T., Di Tommaso, S., Rositi, H., Adrien, J.,Maire, E., Langer, M., Olivier, C., Peyrin, F., and Rogowsky, P.(2015). Fast virtual histology using x-ray in-line phase tomography:Application to the 3D anatomy of maize developing seeds. PlantMethods 11: 55.

Sabelli, P.A., and Larkins, B.A. (2009). The development of endo-sperm in grasses. Plant Physiol. 149: 14–26.

Schmidt, R.J., Burr, F.A., Aukerman, M.J., and Burr, B. (1990).Maize regulatory gene opaque-2 encodes a protein with a “leucine-zipper” motif that binds to zein DNA. Proc. Natl. Acad. Sci. USA 87:46–50.

Schon, M.A., and Nodine, M.D. (2017). Widespread contamination ofArabidopsis embryo and endosperm transcriptome data sets. PlantCell 29: 608–617.

Sekhon, R.S., Lin, H., Childs, K.L., Hansey, C.N., Buell, C.R., deLeon, N., and Kaeppler, S.M. (2011). Genome-wide atlas of tran-scription during maize development. Plant J. 66: 553–563.

Sosso, D., Canut, M., Gendrot, G., Dedieu, A., Chambrier, P.,Barkan, A., Consonni, G., and Rogowsky, P.M. (2012). PPR8522encodes a chloroplast-targeted pentatricopeptide repeat proteinnecessary for maize embryogenesis and vegetative development.J. Exp. Bot. 63: 5843–5857.

Sosso, D., et al. (2015). Seed filling in domesticated maize and ricedepends on SWEET-mediated hexose transport. Nat. Genet. 47:1489–1493.

Sreenivasulu, N., and Wobus, U. (2013). Seed-development pro-grams: A systems biology-based comparison between dicots andmonocots. Annu. Rev. Plant Biol. 64: 189–217.

Suzuki, M., Ketterling, M.G., Li, Q.-B., and McCarty, D.R.(2003). Viviparous1 alters global gene expression patternsthrough regulation of abscisic acid signaling. Plant Physiol. 132:1664–1677.

Trapnell, C., Hendrickson, D.G., Sauvageau, M., Goff, L., Rinn, J.L.,and Pachter, L. (2013). Differential analysis of gene regulation attranscript resolution with RNA-seq. Nat. Biotechnol. 31: 46–53.

UniProt Consortium (2019). UniProt: A worldwide hub of proteinknowledge. Nucleic Acids Res. 47: D506–D515.

Upadhyay, N., Kar, D., Deepak Mahajan, B., Nanda, S., Rahiman,R., Panchakshari, N., Bhagavatula, L., and Datta, S. (2019). Themultitasking abilities of MATE transporters in plants. J. Exp. Bot. 70:4643–4656.

van der Graaff, E., Schwacke, R., Schneider, A., Desimone, M.,Flügge, U.-I., and Kunze, R. (2006). Transcription analysis ofArabidopsis membrane transporters and hormone pathways duringdevelopmental and induced leaf senescence. Plant Physiol. 141:776–792.

Van Lammeren, A.A.M. (1987). Embryogenesis in Zea mays L.: Astructural approach to maize caryopsis development in vivo and

Maize Embryo/Endosperm Interface Transcriptomics 851

in vitro. PhD dissertation (Wageningen, The Netherlands: Wage-ningen University).

Vernoud, V., Hajduch, M., Khaled, A.-S., Depege, N., andRogowsky, P.M. (2005). Maize embryogenesis. Maydica 50:469–483.

Wang, B., Tseng, E., Regulski, M., Clark, T.A., Hon, T., Jiao, Y., Lu,Z., Olson, A., Stein, J.C., and Ware, D. (2016). Unveiling thecomplexity of the maize transcriptome by single-molecule long-read sequencing. Nat. Commun. 7: 11708.

Widiez, T., Ingram, G.C., and Gutiérrez-Marcos, J.F. (2017). Em-bryo-endosperm-sporophyte interactions in maize seeds. In MaizeKernel Development, B. Larkins, ed (Wallingford, UK: CABI), pp.95–107.

Woo, Y.-M., Hu, D.W.-N., Larkins, B.A., and Jung, R. (2001). Ge-nomics analysis of genes expressed in maize endosperm identifiesnovel seed proteins and clarifies patterns of zein gene expression.Plant Cell 13: 2297–2317.

Wu, Y., and Messing, J. (2014). Proteome balancing of the maizeseed for higher nutritional value. Front. Plant Sci. 5: 240.

Yi, F., et al. (2019). High-temporal-resolution transcriptomelandscape of early maize seed development. Plant Cell 31:974–992.

Young, T.E., and Gallie, D.R. (2000). Programmed cell death duringendosperm development. Plant Mol. Biol. 44: 283–301.

Zhan, J., Dannenhoffer, J.M., and Yadegari, R. (2017). Endospermdevelopment and cell specialization. In Maize Kernel Development,B. Larkins, ed (Wallingford, UK: CABI), pp. 28–43.

Zhan, J., Thakare, D., Ma, C., Lloyd, A., Nixon, N.M., Arakaki, A.M.,Burnett, W.J., Logan, K.O., Wang, D., Wang, X., Drews, G.N., andYadegari, R. (2015). RNA sequencing of laser-capture micro-dissected compartments of the maize kernel identifies regulatorymodules associated with endosperm cell differentiation. Plant Cell27: 513–531.

Zhang, S., Wong, L., Meng, L., and Lemaux, P.G. (2002). Similarity ofexpression patterns of knotted1 and ZmLEC1 during somatic andzygotic embryogenesis in maize (Zea mays L.). Planta 215: 191–194.

Zhang, W.Y., Xu, Y.C., Li, W.L., Yang, L., Yue, X., Zhang, X.S., andZhao, X.Y. (2014). Transcriptional analyses of natural leaf senes-cence in maize. PLoS One 9: e115617.

Zheng, Y., and Wang, Z. (2014). Differentiation mechanism andfunction of the cereal aleurone cells and hormone effects on them.Plant Cell Rep. 33: 1779–1787.

Zheng, Y., and Wang, Z. (2015). The cereal starch endosperm de-velopment and its relationship with other endosperm tissues andembryo. Protoplasma 252: 33–40.

Zimmermann, R., and Werr, W. (2005). Pattern formation in themonocot embryo as revealed by NAM and CUC3 orthologues fromZea mays L. Plant Mol. Biol. 58: 669–685.

852 The Plant Cell

DOI 10.1105/tpc.19.00756; originally published online February 21, 2020; 2020;32;833-852Plant Cell

Rogowsky and Thomas WidiezDepège-Fargeix, Eddi Esteban, Asher Pasha, Nicholas J. Provart, Gwyneth C. Ingram, Peter M.

Nicolas M. Doll, Jeremy Just, Véronique Brunaud, José Caïus, Aurélie Grimault, NathalieEndosperm Subdomain Adjacent to the Embryo Scutellum

Transcriptomics at Maize Embryo/Endosperm Interfaces Identifies a Transcriptionally Distinct

 This information is current as of August 10, 2020

 

Supplemental Data /content/suppl/2020/05/13/tpc.19.00756.DC2.html /content/suppl/2020/02/21/tpc.19.00756.DC1.html

References /content/32/4/833.full.html#ref-list-1

This article cites 114 articles, 34 of which can be accessed free at:

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists