Maize reas1 Mutant Stimulates Ribosome Use Efficiency and ... · Maize reas1 Mutant Stimulates Ribosome Use Efﬁciency and Triggers Distinct Transcriptional and Translational Responses1[OPEN]

Maize reas1 Mutant Stimulates Ribosome UseEfficiency and Triggers Distinct Transcriptional andTranslational Responses1[OPEN]

Weiwei Qi2, Jie Zhu2, Qiao Wu2, Qun Wang, Xia Li, Dongsheng Yao, Ying Jin, Gang Wang, Guifeng Wang,and Rentao Song*

Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444,China (W.Q., J.Z., Q.Wu., Q.Wa., X.L., D.Y., Y.J., Ga.W., Gu.W., R.S.); and Coordinated Crop Biology ResearchCenter, Beijing 100193, China (W.Q., Ga.W., Gu.W., R.S.) and National Maize Improvement Center of China,China Agricultural University, Beijing, 100193, China (R.S)

ORCID ID: 0000-0003-1810-9875 (R.S.).

Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects;however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) smallseed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAA-ATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutantallele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, butthe proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryoticinitiation factor 2a and the increased elongation factor 1a level indicate an enhancement of general translational efficiency in dek*cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs.Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinctcellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication andnucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cellgrowth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis.

Ribosomes are organelles that translate genetic in-formation into proteins. A great percentage of totalRNA transcription is devoted to ribosomal RNA syn-thesis, and a great part of RNA polymerase II tran-scription and mRNA splicing are devoted to thesynthesis of ribosomal proteins (Warner, 1999). Ribo-some biosynthesis consumes approximately 80% of acell’s energy (James et al., 2014). In eukaryotes, ribo-some biogenesis begins in the nucleolus with the tran-scription of a large ribosomal precursor RNA that givesrise to the 90S preribosomal particle. Cleavages of the

90S particle generate two subunits: the pre-40S and pre-60S complexes. The pre-40S and pre-60S subunits ma-ture in the nucleolus and nucleoplasm before beingexported to the cytoplasm (Venema and Tollervey,1999; Fromont-Racine et al., 2003; Granneman andBaserga, 2004). Inhibition of ribosome biogenesis causesdevelopmental defects in yeast (Saccharomyces cerevisiae),humans, and plants (Tschochner and Hurt, 2003; Galaniet al., 2004; Ruan et al., 2012).

A great deal of research has revealed that hundreds ofribosomal biogenesis factors contribute to maturation ofthe ribosome in eukaryotes (Tschochner and Hurt, 2003;Henras et al., 2008), including three essential AAA-ATPases: Ribosome export7 (Rix7), Ribosome exportassociated1 (Rea1), and Diazaborine resistance gene1(Pertschy et al., 2007; Kressler et al., 2008, 2012; Ulbrichet al., 2009; Bassler et al., 2010). The Rea1AAA-ATPase isthe best-characterized ATPase in ribosome biogenesisand is conserved from yeast to humans (Bassler et al.,2010; Kressler et al., 2012). Rea1 promotes the stripping ofother biogenesis factors from the pre-60S particle in thenucleolus and nucleoplasm (Ytm1-Erb1-Nop7 and Rsa4)prior to the export of the large ribosomal subunit to thecytoplasm (Bassler et al., 2010). However, there is not acomprehensive understanding of cellular responses tothe impaired large ribosomal subunit export.

The regulation of mRNA translation is a critical fea-ture of gene expression in eukaryotes (Bailey-Serres,

1 This work was supported by the Major Research Plan of theNational Natural Sciences Foundation of China (grant nos.91335208 and 31425019) and the Ministry of Science and Technologyof China (grant no. 2014CB138204).

2 These authors contributed equally to the article.* Address correspondence to [email protected] author responsible for distribution of materials integral to the

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

R.S., W.Q., and J.Z. designed the experiments; J.Z., W.Q., Q.Wu.,Q.Wa., X.L., D.Y., and Y.J. performed the experiments; W.Q., J.Z.,Ga.W., Gu.W., and R.S. analyzed the data; W.Q. and R.S. wrote thearticle.

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1999). Previous studies highlight the importance oftranslational control in determining protein abundance,underscoring the value ofmeasuring gene expression atthe level of translation. Mechanisms that underlie dif-ferential mRNA translation are likely to involve nu-cleotide sequence features and the phosphorylationstatus of initiation factors (Bailey-Serres and Dawe,1996; Pop et al., 2014). Transcriptome and translatomeanalyses of the cellular response to heat shock, cell cyclearrest, and mating pheromone in Saccharomyces cer-evisiae (Preiss et al., 2003; Serikawa et al., 2003; MacKayet al., 2004), the hypoxia response of HeLa cells (Blaiset al., 2004), and the drought and oxygen deprivation re-sponses in Arabidopsis (Arabidopsis thaliana; Kawaguchiet al., 2004; Branco-Price et al., 2005) have shown theimportance of translational regulation. These researchersinvestigated the correlation between total and polyri-bosome (polysome)-bound mRNA accumulation andprovided extensive evidence of variation in the transla-tional regulation of individual mRNAs. These studiesshowed that mRNAs differ in their association withpolysomes under different circumstances, and geneexpression can be regulated at the translational levelwithout a change in mRNA abundance.

Maize (Zea mays) is especially well suited for geneticstudies, partly because of the feasibility to generate awide range of easily observable phenotypes (Neufferand Sheridan, 1980). Many kernel mutants are known(Neuffer et al., 1968), among which one class is defectivekernel (dek) mutants (Neuffer and Sheridan, 1980). dekmutants are a good resource to investigate seed devel-opment. For example, Dek1 encodes a large membraneprotein of the calpain gene superfamily (Lid et al.,2002). In dek1mutants, embryogenesis is blocked, whilethe endosperm lacks the aleurone layer and is chalky(Becraft et al., 2002). Other dek mutants offer opportu-nities to investigate many basic biological processes,because embryo formation is the first developmentalprocess after fertilization. Such defects in basic biolog-ical processes create visible phenotypes during kerneldevelopment.

In this study, we characterized dek*, a novel mutantwith small kernels and delayed development of theembryo, endosperm, and seedling. We report the map-based cloning of Dek* and demonstrate that it encodesRea1 in maize. dek* is a weak mutant allele that onlypartly represses the maturation and export of the 60Sribosomal subunit. Taking advantage of this mutantallele, we were able to obtain comprehensive informa-tion about the cellular responses to impaired 60S ribo-somal subunit biogenesis.

RESULTS

dek* Produces Small Kernels with Delayed Development

The dek* mutant was isolated from an opaque mutantstock obtained from the Maize Genetic Stock Center. Itwas crossed to the W64A inbred line to produce anF2 population that displayed a 1:3 segregation of dek

(dek*/dek*) andwild-type (+/+ and dek*/+) phenotypes(Fig. 1, A and B). At 15 DAP, homozygous dek* kernelsexhibited a small, vague phenotype (Fig. 1A), and ma-ture kernels were small and shrunken (Fig. 1B). The100-kernel weight of dek* was nearly 39.5% less thanthat of the wild type (Fig. 1C), but there was no sig-nificant difference in the total protein and zein contents(Fig. 1D; Supplemental Fig. S1), although there wasa slight increase in the amount of nonzeins (13.5%;Fig. 1D). Among zein proteins, the 22-kD a-zeinswere relatively more abundant in dek* endosperms(Supplemental Fig. S1).We found no obvious differencein total starch content and the percentage of amylose indek* and wild-type endosperms (Supplemental Fig. S2).We analyzed soluble amino acids to determine if theslight increase of nonzeins in dek* altered their compo-sition. The results showed that the amount of Lys wasmost significantly increased (23.1%) due to the slightincrease of nonzein content (Fig. 1E), for zeins lack Lysresidues (Mertz et al., 1964).

Wild-type and dek* kernels of 15 and 18 DAP wereanalyzed by light microscopy to compare their develop-ment. Longitudinal sections of the embryos indicated thatdevelopment of the plumule and seminal was delayedmore than 3 d in dek* compared with the wild type (Fig.1F). To investigate endospermdevelopment,we observed15- and 18-DAP immature endosperm cells using opticalmicroscopy. The endosperm cells of dek* kernels were lesscytoplasmic dense with fewer starch granules comparedwith wild-type kernels of the same stage, also indicatingmore than a 3-d delay in development (Fig. 1G).

At 4 and 7 d after germination (DAG), seedlings ofdek* showed a 3-d developmental delay compared withthe wild type (Fig. 1H). By 4 DAG, wild-type seedlingshad two leaves, one completely expanded and the otheremerging; the dek* seedlings had only one leaf at thisstage. The 7-DAGwild-type seedlings had three leaves,while the dek* seedlings had only two leaves. Theheading stage of the dek* plant was delayed approxi-mately 15 d comparedwith the wild type, and its heightwas only 50% of the wild type (Supplemental Fig. S3).These results demonstrated that the growth and de-velopment of dek* kernels and seedlings is delayedcompared with the wild type.

Positional Cloning of Dek*

Genetic fine-mapping of Dek* was carried out withthe F2 mapping population, and the Dek* gene wasplaced between the simple sequence repeat markersmmc0241 and umc2162 on the long arm of chromosome6 (Fig. 2A). After characterizing a mapping populationof 864 individuals, Dek* was mapped between theself-created simple sequence repeat markers 153.7M-2(19 recombinants) and 155.1M-1 (29 recombinants).Additional markers InDel438, InDel428, SNP064, andSNP165 were developed, and the Dek* gene was even-tually placed between SNP064 (one recombinant) andSNP165 (two recombinants), a region encompassing aphysical distance of 101.6 kb (Fig. 2A).

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Nucleotide sequence analysis within this region iden-tified 10 predicted open reading frames with gene modelinformation (GRMZM2G405052, GRMZM2G387038,GRMZM5G873561, GRMZM5G807823, GRMZM2G361064,GRMZM5G892685, GRMZM2G059268, GRMZM2G059278,GRMZM2G323939, and GRMZM2G128315). Expressionanalysis revealed no expression of GRMZM5G892685,

GRMZM2G059268, and GRMZM2G059278 basedon reverse transcription-PCR and EST information(http://www.maizegdb.org/); consequently, thesethree might be pseudogenes. DNA sequence analysisrevealed that GRMZM2G405052, GRMZM2G387038,GRMZM5G873561, and GRMZM5G807823, togetherwith GRMZM2G092001 and GRMZM2G149586,

Figure 1. Phenotypic features of maize dek*/reas1-refmutants. A, A 15-d after pollination (DAP) F2 ear of dek*3W64A and randomlyselected 15-DAP dek* andwild-type (WT) kernels in a segregated F2 population. The red arrow identifies the dek* kernel. Bar = 5mm. B,Mature F2 ear of dek*3W64A and randomly selected mature dek* and wild-type kernels in a segregated F2 population. The red arrowidentifies the dek* kernel. Bar = 5 mm. C, Comparison of 100-grain weight of randomly selected mature dek* and wild-type kernels in asegregated F2 population. Values are means with SE; n = 3 individuals (***, P, 0.001, Student’s t test). D, Comparison of total, zein, andnonzeinproteins fromdek* andwild-typemature endosperm.Themeasurementsweredonepermgof driedendosperm.Values aremeanswith SE; n= 3 individuals (ns, not significant; **, P, 0.01, Student’s t test). E, Soluble amino acidswith different contents in dek* andwild-typemature endosperm. Values are means with SE; n = 3 individuals (*, P, 0.05; **, P, 0.01; ***, P, 0.001, Student’s t test). F, Paraffinsections of 15- and 18-DAP dek* and wild-type embryos. Bars = 200 mm. G, Microstructure of developing endosperms of dek* and thewild type (15 and18DAP). SG, Starch granule. Bars =100mm.H,Phenotypes ofdek* andwild-type seedlings (4 and7DAG). Bars =5cm.

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Characterization of the Maize reas1 Mutant


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which are upstream of the candidate region, producedone huge transcript that was identified as candidateGene1. There is a single-nucleotide polymorphism inGene1 resulting in an amino acid replacement betweenthe alleles of dek* and the wild type. GRMZM2G361064,GRMZM2G323939, and GRMZM2G128315 were iden-tified as candidate Gene2, Gene3, and Gene4, re-spectively; however, their consideration for Dek* waseliminated due to no sequence differences between al-leles in dek* and the wild type (Fig. 2A). Therefore,Gene1 appeared to be the best candidate for the Dek*locus.

Dek* Encodes the 60S-Specific Ribosome BiogenesisFactor Rea1

The genomic DNA sequence of candidate Gene1spans approximately 50 kb and produces a hugetranscript containing a 16,278-bp coding sequence(Fig. 2B). Sequence data for this gene have been de-posited in GenBank (http://www.ncbi.nlm.nih.gov/)as accession number KP137367. Gene1 encodes anapproximately 600-kD protein of 5,425 amino acids.BLASTP searches of GenBank indicated that Gene1encodes a ribosome biogenesis factor, AAA-ATPaseRea1, with several conserved domains in maize (Fig.2B). And we named it ZmReas1. ZmReas1 containsdifferent kinds of molecular domains: a weakly con-served N-terminal region, a dynein-like tandem arrayof six AAA-type ATPase domains (Neuwald et al.,1999), a large linker, a D/E-rich region, and a metal

ion-dependent adhesion site (MIDAS) domain (Fig.2B). Rea1 promotes the release of Ytm1, which asso-ciates with nucleolar pre-60S particles, and later alsopromotes the release of Rsa4, which associates withnucleoplasmic pre-60S particles via the MIDAS-MIDAS-interacting domain using the mechanical force createdby the ATPase ring domain for the export of the largeribosomal subunit to the cytoplasm (Ulbrich et al., 2009;Bassler et al., 2010). The mutation in the dek* allele ofZmReas1 is a single-nucleotide polymorphism at codon2,359 of ZmReas1, which results in Ala (GCC) beingreplaced by Val (GTC; Fig. 2B). This mutation alters thehighly conserved region between the dynein-like array ofsix AAA-type ATPases and the large linker, which couldaffect the transduction of the mechanical force created bythe ATPase ring domain to the large tail for release of theribosome biogenesis factors.

To confirm ifZmReas1 is theDek* gene, we carried outan allelism test with a Mu-induced mutant of ZmReas1(Fig. 2, B and C). A UniformMu insertion mutant (reas1-Mu) stock for GRMZM2G092001 was obtained fromthe Maize Genetics Stock Center. This mutant has aMutator-8 insertion after the fourth nucleotide of theZmReas1 coding sequence and is not viable (Fig. 2B).The allelism test was done by crossing dek* F1 (dek*/+)and reas1-Mu F1 (reas1-Mu/+). The kernel phenotypesin the F2 ears displayed a 1:3 segregation of 82 dek(dek*/reas1-Mu) and 249 wild-type phenotype kernels(Fig. 2C), indicating that reas1-Mu cannot complementdek*. Therefore, Gene1 (ZmReas1) is indeed the Dek*gene. We hence named dek* as reas1-ref.

Figure 2. Map-based cloning and identification of Reas1A, TheDek* locuswasmapped to a 101.6-kb region betweenmolecularmarkers SNP064 and SNP165 on chromosome 6, which contained four candidate genes. For primer information, seeSupplemental Table S3. B, Protein structure of ZmReas1 andmutation sites in the ZmReas1 gene. C, Heterozygous reas1-ref/dek*and reas1-Mu were used in an allelism test because homozygous reas1-Mu was lethal. Top, heterozygous reas1-ref 3 heterozygousreas1-Mu; middle, heterozygous reas1-Mu3 heterozygous reas1-ref; bottom, heterozygous reas1-Mu3 heterozygous reas1-Mu. Thered arrows identify the reas1 kernel. D, qRT-PCR comparing the expression levels of ZmReas1 in 15- and 18-DAP reas1-ref and wild-type (WT) kernels. Ubiquitin was used as an internal control. Values are means with SE; n = 3 individuals (***, P, 0.001, Student’s ttest). E, Dot immunoblot comparing the accumulation of ZmReas1 protein in 15- and 18-DAP reas1-ref and wild-type kernels. The720-, 360-, and 180-ng 15- and 18-DAP reas1-ref and wild-type kernel proteins were subjected to immunoblot analysis with anti-bodies against ZmReas1.


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Maize endosperm is a triploid tissue with two ma-ternal and one paternal genomes. Themutant kernels inreas1-ref/+ (maternal) 3 reas1-Mu/+ (paternal) F2 earare small and shallow, similar to homozygous reas1-refkernels. The mutant kernels in reas1-Mu/+ (maternal) 3reas1-ref /+ (paternal) F2 ears display an even more se-vere phenotype with dramatically shrunken kernels. Themutant kernels from reas1-Mu/+ selfing are nonviable(Fig. 2C). Thus, results of the allelism test show that reas1-ref is a weak allele compared with reas1-Mu, which has alethal phenotype.To examine Reas1 mRNA expression in reas1-ref, we

performed quantitative reverse transcription (qRT)-PCRwith the total RNA extracted from 15- and 18-DAPmutant and wild-type kernels. Surprisingly, mRNAexpression of Reas1 was significantly up-regulated inreas1-ref (Fig. 2D). Because ZmReas1 is too large (ap-proximately 600 kD) to perform a regular western-blotanalysis, we used dot-immunoblot analysis on quanti-fied and gradient-diluted total protein samples withReas1 specific antibody to detect its existence in 15- and18-DAP reas1-ref and wild-type kernels (Fig. 2E). Theresults demonstrated that Reas1 is present in reas1-refand accumulates in reas1-ref at normal levels, but itmight be only partly functional.

Reas1 Is Highly Conserved in Different Organisms and IsConstitutively Expressed in Maize

Rea1 was first identified as a component of pre-60Sribosome complex in yeast and is conserved from yeastto humans (Bassler et al., 2001, 2010; Kressler et al.,2012). We constructed a phylogenetic tree on the basisof the ZmReas1 full-length protein sequence and Rea1protein sequences from Brachypodium distachyon, Triti-cum urata, Oryza sativa, Setaria italica, Arabidopsis,Populus trichocarpa, Glycine max, Dictyostelium dis-coideum, Monodelphis domestica, Saprelegnia diclina,Mortierella verticillata, and Saccharomyces cerevisiae. Theresults suggest that ZmReas1 is highly conservedwith theRea1 proteins in other plants as well as the Rea1 proteinsof yeast, mammals, and microorganisms (Fig. 3A).Quantitative RT-PCR analysis revealed that ZmReas1

is expressed in a broad range ofmaize tissues, includingsilk, tassel, ear, root, husk, stem, leaf, and kernel (Fig.3B). During kernel development, expression of Reas1occurs before 5 DAP and continues later than 25 DAP(Fig. 3C). Dot-immunoblot analysis on quantified andgradient-diluted total nuclear and cytoplasmic proteinsdetected Reas1 in these subcellular fractions, and it waspredominantly found in the nuclear fraction, consistentwith Reas1 localization in the nucleus (Fig. 3D).

reas1-ref Affects the Biogenesis of 60S Ribosomal Subunits

To investigate the effect of reas1-ref on ribosomalsubunit biogenesis and the formation ofmonosome andpolysome complexes, polysome profiles of 15-DAPreas1-ref and wild-type kernel extracts were analyzedby 15% to 45% (w/v) Suc gradient centrifugation. Two

independent biological replicates were performed. Thisanalysis revealed a significant reduction of 60S ribo-somal subunits, as compared with 40S ribosomal sub-units, in the mutant (Fig. 4A). To compare the levels ofmonosomes and polysome complexes, calculation ofthe peak areas of A254 revealed that about 40.2% of theribosomes in wild-type kernel extracts were in poly-somes, while the level of polysome complexes in reas1-ref kernel extracts was 57.2% (Fig. 4A). Thus, there are1.4-fold greater polysomes/total ribosomes in reas1-ref.The decrease in 60S subunits and the increase in poly-somes are consistent with the inhibition of large ribo-somal subunit export and the promotion in initiation ofprotein synthesis as a consequence of down-regulatedribosome biogenesis in reas1-ref.

To confirm that maturation and export of 60S subu-nits are reduced in reas1-ref, immunoblot analysis with60S and 40S subunit antibodies was performed on nu-clear and cytoplasmic fractions from 15- and 18-DAPreas1-ref or wild-type kernels. Nuclear and cytoplasmicfractions were subjected to immunoblot analysis withantibodies against Bip (cytoplasm marker) and histone(nucleus marker). Tubulin and TATA box-bindingprotein (TBP) served as sample loading controls of cy-toplasmic and nuclear proteins, respectively. The levelof eukaryotes 60S ribosomal protein L13 eRPL13 wasexamined using an L13-specific antibody. L13 proteinwas markedly decreased in both the nuclear and cyto-plasmic fractions of reas1-ref compared with wild-typekernels (Fig. 4B). The level of eRPS14 was also exam-ined using a specific antibody, and its content was thesame in both the nuclear and cytoplasmic fractions ofreas1-ref andwild-type kernels (Fig. 4B).Meanwhile, wealso observed a slight decrease of histone protein in thereas1-ref nuclear fraction (Fig. 4B).

A reduction of 60S ribosomal subunits in the cyto-plasm of 15- and 18-DAP reas1-ref and wild-type en-dosperms was also observed by transmission electronmicroscopy (TEM) analysis. There were fewer ribo-somes on rough endoplasmic reticulum and therough endoplasmic reticulum around protein bodies(Supplemental Fig. S4). Nucleolus stress due to ribo-somal failure alters the morphology and increases thesurface area of nucleolus in humans (Bailly et al., 2015).This stress, which misshapes and expands the nucleo-lus, was also observed in reas1-ref endosperm by TEM(Fig. 4C). All these data are consistent with a biogenesisdefect of 60S subunits and demonstrates that it is spe-cific to the 60S maturation and export pathway.

reas1-ref Affects the Transcription of Ribosome Biogenesis,Translational Elongation, and Nucleosome-Related Genes

We compared the transcript profiles of 15-DAP reas1-ref and wild-type endosperm using RNA sequencing(RNA-seq). Among the 45,730 gene transcripts detectedby RNA-seq, significantly differentially transcribedgenes (DTGs) were identified as those with a thresholdfold change greater than 2 and P , 0.05. Based on thiscriterion, 2,076 genes showed significantly altered






expression between reas1-ref and the wild type. Therewere 1,518 genes with increased transcription, while558 genes showed decreased transcription.

Within the 2,076 DTGs, 39.9% could be functionallyannotated (annotations were found using BLASTN andBLASTX analyses against the GenBank (http://www.

ncbi.nlm.nih.gov/) database. Gene Ontology (GO;http://bioinfo.cau.edu.cn/agriGO/) and Kyoto En-cyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) pathway analysis indicated that 828DTGs were mostly related to four GO terms: GO: 0005840(ribosome; P = 2.34E-130), GO: 0006414 (translational

Figure 3. Phylogenetic analysis, expression pattern, and subcellular localization of ZmReas1 A, Phylogenetic relationships ofZmReas1 and its homologs. Maize Reas1 and identified Rea1 proteins in B. distachyon, T. urata,O. sativa, S. italica, Arabidopsis,P. trichocarpa, G. max, D. discoideum,M. domestica, S. diclina,M. verticillata, and S. cerevisiae were aligned by the MUSCLEmethod in the MEGA 5.2 software package. The phylogenetic tree was constructed using MEGA 5.2. The numbers at the nodesrepresent the percentage of 1,000 bootstraps. B, RNA expression level of ZmReas1 in various tissues. Ubiquitin was used as aninternal control. Representative results from two biological replicates are shown. For each RNA sample, three technical replicateswere performed. Values are means with SE; n = 6 individuals. C, Expression profiles of ZmReas1 during maize kernel develop-ment. Ubiquitin was used as an internal control. Representative results from two biological replicates are shown. For each RNAsample, three technical replicates were performed. Values are means with SE; n = 6 individuals. D, Dot-immunoblot analysis ofZmReas1 protein accumulation. Reas1 is predominantly associatedwith the nuclear protein fraction. The 720-, 360-, and 180-ngnuclear (histone as nuclear marker) and cytoplasmic (Bip as cytoplasm marker) fraction proteins were subjected to immunoblotanalysis with antibodies against ZmReas1.


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elongation; P = 2.30E-17), GO: 0000786 (nucleosome;P = 8.22E-29), and GO: 0045735 (nutrient reservoir activ-ity; P = 6.47E-33). This analysis is illustrated in Figure 5Aand Supplemental Table S1.Ninety-eight DTGs classified to GO: 0005840 (ribo-

some) could be divided into three categories: small ribo-somal subunit proteins (e.g. eRPS6 [GRMZM5G851698]and eRPS13 [GRMZM2G130544]), large ribosomal subu-nit proteins (e.g. eRPL14 [GRMZM2G168330] andeRPL18 [GRMZM2G030731]), and ribosome biogenesisfactors. Transcription of all the genes related to ribosomebiogenesis was increased in reas1-ref endosperm. reas1-refalso has a strong impact on translational elongation.

The 20 DTGs involved in GO: 0006414 (translationalelongation) could be divided into two categories: 60Sacidic ribosomal proteins (e.g. eRPLP0 [GRMZM2G066460]and eRPLP1 [GRMZM2G157443]) and translation elonga-tion factors (e.g. eEF1a [GRMZM2G151193] and eEF1b[GRMZM2G122871]). These genes were also up-regulated.Fifty-twoDTGs related to GO: 0000786 (nucleosome) couldbe divided into two categories: histones (e.g. H2A[GRMZM2G056231] and H2B [GRMZM2G401147]) andnucleosome assembly protein (GRMZM2G176707). Tran-scription of these genes, which are related to nucleosomeassembly and the cell cycle,wasmarkedly induced in reas1-ref endosperm. DTGs involved in GO: 0045735 (nutrient

Figure 4. The production of mature 60S subunits isreduced in reas1-ref kernels. A, Analysis of ribosomeprofiles (A254) was performed by sedimentation cen-trifugation in 15% to 45% Suc density gradients: 40S,60S, and 80S ribosomes and polysomes are indi-cated. B, Immunoblot analysis of ribosome proteinsaccumulated in nuclear and cytoplasmic fractions.Nuclear and cytoplasmic fraction proteins of 15- and18-DAP reas1-ref and wild type (WT) kernels weresubjected to immunoblot analysis with antibodiesagainst eRPL13 (60S ribosomal subunit marker),eRPS14 (40S ribosomal subunit marker), Bip (cyto-plasm marker), Histone (nuclear marker), Tubulin(cytoplasm sample loading control), and TBP (nuclearsample loading control). C, Ultrastructure of devel-oping endosperms of the wild type and reas1-ref (15DAP) for nucleus observation. There was nucleolusstress, as shown by the expended nucleolus in reas1-ref.NL, Nucleolus; NP, nucleoplasm; SG, starch granule.The nucleolus size/nucleus size measurements weredone on TEM results. Values are means with SE; n = 10individuals (**, P, 0.01, Student’s t test). Bars = 2 mm.






reservoir activity) were storage proteins, including22-kD a-zein (GRMZM2G346897) and 19-kD a-zein(GRMZM2G059620); that is, these genes were down-regulated. To validate the differences observed by RNA-seq, we performed qRT-PCR on the most significantDTGs from each GO category, and the results confirmedsimilar differences of mRNA accumulation (Fig. 5B).

reas1-ref Exhibits Uncoordinated Expression of DistinctGroups of Genes at the Translational Level

The increase in polysomes in reas1-ref indicated pro-motion in the initiation of protein synthesis in responseto down-regulated ribosome biogenesis. The mecha-nisms that underlie differences of mRNA translationinvolve sequence features of individual mRNAs andthe phosphorylation status of translation initiationfactors (Bailey-Serres and Dawe, 1996). General ControlNonderepressing kinase2 (GCN2) was reported tophosphorylate eukaryotic initiation factor 2a (eIF2a) todown-regulate translation (Zhang et al., 2008). We firstmeasured the phosphorylation levels of eIF2a in 15-and 18-DAP reas1-ref and wild-type cytoplasm byprotein gel-blot analysis with P-eIF2a and eIF2a (totaleIF2a as a control) antibodies. Compared with the wildtype, eIF2a in reas1-ref was significantly less phos-phorylated, while the eIF2a protein level was not al-tered (Fig. 6A). The level of eEF1a protein in reas1-refand wild-type cytoplasm was examined using specificantibody. eEF1a was markedly increased in reas1-ref

(Fig. 6A). These results indicated that the initiation andelongation of translation are promoted in reas1-ref.

The level of an mRNA in polysomes reflects itstranslation (Branco-Price et al., 2005). To examine theeffects of reas1-ref on the translational regulation of in-dividual mRNAs, we evaluated the amount of RNAin polysomes, relative to the total amount of transcriptin 15-DAP reas1-ref and wild-type kernels. Kernel extractswere centrifuged (170,000g) to obtain a polysome pelletfor comparing total extract and polysome-bound RNAsamples by RNA-seq analysis. The polysome-bound-to-total RNA ratios in 15-DAP reas1-ref and wild-typekernels were 36.4% and 25.6%, respectively. Conse-quently, there was a 1.4-fold increase of polysome-bound-to-total RNA in reas1-ref, which is consistentwith the polysome complex/total ribosome A254 by ri-bosome profile analysis.

Within the 30,188 gene transcripts detected byRNA-seq,significantly differentially translated RNAs (DTRs) wereidentified as those with a 2nP/T (normalized polysome-bound/total) 3100% (see “Materials and Methods”) between reas1-refand the wild type, fold change greater than 2 or P, 0.5.Based on this criterion, 1,802 genes showed significantlyincreased translation in reas1-ref compared with the wildtype, while 2,959 genes showed decreased translation. Toconfirm the differences between wild-type and reas1-refendosperm observed by RNA-seq, we performed qRT-PCR on the most significantly increased or decreasedDTRs selected from each category, and the results wereconsistent (Fig. 6, B and C). We also performed

Figure 5. GO classification for genes with altered expression in reas1-ref kernels. A, The most significantly related GO terms ofthe 828 functional annotatedDTGs. The significance and number of genes classifiedwithin eachGO term are shown. B, qRT-PCRconfirmation of DTGs associated with each category, including small ribosomal subunit proteins (GRMZM5G851698,GRMZM2G120432, GRMZM2G130544, GRMZM2G156110, and GRMZM2G151252); large ribosomal subunit proteins(GRMZM2G132968, GRMZM2G100403, GRMZM2G168330, GRMZM2G030731, and GRMZM2G010991); ribosome bio-genesis factors (GRMZM2G063700, GRMZM2G110233, and GRMZM2G468932); 60S acidic ribosomal proteins(GRMZM2G157443 and GRMZM2G077208); translation elongation factors (GRMZM2G151193, GRMZM2G153541,GRMZM2G122871, and GRMZM2G029559); histones (GRMZM2G056231, GRMZM2G401147, GRMZM2G078314,GRMZM2G479684, and GRMZM2G164020); nucleosome assembly protein (GRMZM2G176707); and nutrient reservoir ac-tivity (GRMZM2G346897, GRMZM2G059620, and GRMZM2G138727). Ubiquitin was used as an internal control. Values aremeans with SE; n = 6 individuals (***, P , 0.001, Student’s t test). WT, Wild type.


Qi et al.



puromycin treatment for the release of polysome asa negative control (Supplemental Fig. S5). No signifi-cant difference of sequence features was observed be-tween the up-regulated and down-regulated DTRs(Supplemental Table S2).Within the increased DTRs, 687 could be functionally

annotated. GO analysis indicated that these RNAs aremostly related to three GO terms, GO: 0045449 (regu-lation of transcription; P = 1.33E-03), GO: 0045735(nutrient reservoir activity; P = 3.51E-12), and GO:0006414 (translational elongation; P = 1.17E-07). For thedecreased DTRs, 601 could be functionally annotated,belonging to GO: 0006334 (nucleosome assembly; P =6.59E-10), GO: 0006260 (DNA replication; P = 1.18E-03),and GO: 0033279 (ribosomal subunits; P = 1.16E-03).Ten most strongly up-regulated or down-regulatedDTRs of each classification are illustrated in Table I,and all DTRs are shown in Supplemental Table S2.Transcriptional factors (e.g. NAC domain transcrip-

tion factors and MADS box transcription factors) andtranslation elongation-related genes had markedlyhigher translation levels in reas1-ref. Although the ex-pression of zeins was down-regulated in reas1-ref (Fig.5), surprisingly, their translation level was increasedsignificantly (Table I; Supplemental Table S2). Therewas significant overlap (P = 2.23E-16, x2 test) for zeinsbetween transcriptional down-regulated genes andtranslational up-regulated genes (Fig. 6E). Meanwhile,although the expression of histone RNAs was up-

regulated in reas1-ref (Fig. 5), their translation levelwas dramatically reduced. There is also significantoverlap (P = 1.57E-14, x2 test) for histones betweentranscriptional up-regulated genes and translationaldown-regulated genes (Fig. 6E). DNA replication-related genes (e.g. DNA polymerase subunits andminichromosome maintenance proteins [MCMs]) hadlower translation levels in reas1-ref. Histones and DNAreplication-related genes are both related to nucleo-some assembly and the cell cycle. Although the tran-scription of ribosomal subunit proteins is up-regulatedin reas1-ref (Fig. 5), their translation level is dramaticallydown-regulated. There is overlap between transcrip-tional up-regulated genes and translational down-regulated genes for ribosomal proteins (Fig. 6E). Theseresults demonstrate that the transcriptional and trans-lational regulation of individual genes respondingto reduced 60S ribosome exportation is not alwaysconsistent.

We measured the level of 22-kD a-zeins in 470 and1,190 ng of total proteins of 15-DAP reas1-ref and wild-type kernels, respectively, by protein gel-blot analysiswith 22-kD a-zein antibodies. Compared with the wildtype, 22-kD a-zeins in reas1-ref were increased signifi-cantly. Meanwhile, there was no effect on 19-kD a-zeincontent (Fig. 6D). Increased eEF1a protein content (Fig.6A) and lower protein content of histone (Fig. 4B) inreas1-ref also confirmed their increased or decreasedtranslation level.

Figure 6. Induced general translation efficiency and specific regulation of translation of individual mRNAs in reas1-ref kernels.A, Immunoblot comparing the phosphorylated (P) eIF2a accumulation in wild-type (WT) and reas1-ref kernels (15 and 18 DAP).Anti-eIF2a was used as a control immunoblot comparing the accumulation of eEF1a in wild-type and reas1-ref kernels at thesame stage. Anti-TUB was used as a sample loading control. B and C, qRT-PCR confirmation of DTRs with increased(GRMZM2G081930, GRMZM2G007038, and GRMZM2G110509) or decreased (GRMZM2G084195, GRMZM2G154267, andGRMZM2G021069) translation levels. Samples were mRNA preparations from the polysome fractions. Ubiquitin was used as aninternal control. Values are meanswith SE; n = 6 individuals (*, P, 0.05; **, P, 0.01, Student’s t test). D, Immunoblot comparingthe accumulation of 22- and 19-kD a-zein in 470 and 1,190 ng of total protein of 15-DAP reas1-ref and wild-type kernels byprotein gel-blot analysis with 22- and 19-kD a-zein-specific antibodies. E, Overlap between transcriptional up-regulated genesand translational down-regulated genes for ribosomal proteins and histones, and overlap between transcriptional down-regulatedgenes and translational up-regulated genes for zeins.









Table I. Ten most strongly up-regulated or down-regulated DTRs of each GO classification

GO Identifier P Gene DescriptionPolysome/Total in

the Wild Type

Polysome/Total

in reas1

Fold

Change

% of totalGenes with increased ratio of polysome-bound mRNAGO: 0045449, regulation of

transcription1.33E-03 GRMZM2G081930 NAC1 0.1437 0.6978 4.86

GRMZM2G167018 NAC domain transcription factor 0.0336 0.3461 10.31GRMZM2G134717 NAC domain transcription factor 0.0291 0.2145 7.36GRMZM2G170079 BZIP-type transcription factor 0.0165 0.1979 12.03GRMZM5G812272 WRKY DNA-binding domain

superfamily protein0.0106 0.1038 9.76

GRMZM2G327059 BEL1-related homeotic protein 0.0316 0.2861 9.05GRMZM2G021339 Leu zipper domain protein 0.0158 0.1272 8.07GRMZM2G126239 Homeobox-Leu zipper protein

ATHB-40.0142 0.2754 19.38

GRMZM2G087741 Homeobox protein liguleless3 0.0480 0.3018 6.29GRMZM5G809195 IAA14-auxin-responsive Aux/IAA

family member0.0451 0.4536 10.06

GO: 0045735, nutrientreservoir activity

3.51E-12 GRMZM2G346897 22-kD a-zein 0.2875 0.7691 2.68

GRMZM2G353272 22-kD a-zein 0.2442 0.9829 4.03GRMZM2G044152 22-kD a-zein 0.2126 0.7821 3.68GRMZM2G397687 22-kD a-zein 0.2153 0.8910 4.14GRMZM2G053120 22-kD a-zein 0.2127 0.9571 4.51GRMZM2G008341 Zein-a 19-kD z1A 0.2862 0.9428 3.29GRMZM2G353268 Zein-a 19-kD z1A 0.2504 0.8631 3.45AF546188.1_FG003 Zein-a 19-kD z1B 0.2120 0.7470 3.52AF546188.1_FG007 Zein-a 19-kD z1B 0.2578 0.8318 3.23AF546187.1_FG007 Zein-a 19-kD z1D 0.2429 0.9263 3.81

GO: 0006414, translationalelongation

1.17E-07 GRMZM5G859846 Elongation factor Tu 0.0476 0.1744 3.67

GRMZM2G007038 Elongation factor Tu 0.2158 0.6634 3.07GRMZM2G407996 Elongation factor Tu 0.3131 0.6392 2.04GRMZM2G110509 Elongation factor 1a 0.1984 0.4218 2.13GRMZM2G151193 Elongation factor 1a 0.2001 0.4162 2.08GRMZM2G001327 Elongation factor 1a 0.2248 0.4732 2.11

Genes with reduced ratio of polysome-bound mRNAGO: 0006334, nucleosome

assembly6.59E-10 GRMZM5G883764 Histone H2A 0.5549 0.0717 0.13

GRMZM2G355773 Histone H3.2 0.9458 0.0945 0.09GRMZM2G447984 Histone H3.2 0.8418 0.1126 0.13GRMZM2G130079 Histone H3.2 0.6460 0.0847 0.13GRMZM2G349651 Histone H4 0.7069 0.0850 0.12GRMZM2G073275 Histone H4 0.5894 0.0784 0.13GRMZM2G479684 Histone H4 0.9550 0.1012 0.11GRMZM2G084195 Histone H4 0.3357 0.0266 0.08GRMZM2G421279 Histone H4 0.9435 0.1030 0.11GRMZM2G149178 Histone H4 0.5823 0.0750 0.13

GO: 0006260,DNA replication

1.18E-03 GRMZM5G825512 Origin recognition complexsubunit 6

0.4301 0.1093 0.25

GRMZM5G872710 DNA polymerase 0.4674 0.1236 0.26GRMZM2G154267 DNA polymerase «-subunit 2 0.7544 0.0657 0.09GRMZM2G100639 DNA replication licensing factor

MCM3 homolog 20.6059 0.0796 0.13

GRMZM2G117238 Origin recognition complexsubunit 2

0.6233 0.1285 0.21

GRMZM2G162445 Minichromosome maintenancecomplex protein family

0.8304 0.0891 0.11

GRMZM2G086934 Replication protein A 70-kDDNA-binding subunit

0.5513 0.1185 0.22

GRMZM2G021069 Minichromosome maintenanceprotein

0.7020 0.0859 0.12

(Table continues on following page.)


Qi et al.



reas1-ref Inhibits Cell Proliferation and Cell Growth

The synthesis of nucleosome assembly proteins relatedto the cell cycle transition is markedly reduced in reas1-ref(Table I; Fig. 6). This mutant exhibits a slow-growthphenotype for both kernels and seedlings. There was amore than a 3-d delay in endospermdevelopment (Fig. 1).Endoreduplication is a general feature of endosperm de-velopment in maize, involving replication of the nucleargenome without cell division and leading to elevatednucleic acid content (Sabelli and Larkins, 2009). Endore-duplication includes only G1 and S phases, which is dif-ferent from the mitotic cell cycle (G1-S-G2-M phases).Flow cytometry analysis of 15-DAP reas1-ref and wild-

type endosperms showed endoreduplicated nuclei withC values of 12C or greater, accounting for 18.1% of theDNA in 15-DAP endosperm of reas1-ref and 22.2% of theDNA in 15-DAP wild-type endosperm (Fig. 7A). At 18DAP, there were 19.3% and 24.2% endoreduplicated nu-clei with C values of 12C or greater in reas1-ref and wild-type endosperm, respectively (Fig. 7B). The mitotic cellcycle was also assessed in 7-DAG seedlings by flow cy-tometry. The results showed that 63.3% of the nuclei have2C DNA content in reas1-ref 7-DAG seedlings, while45.1% of the nuclei in the wild-type seedlings have 2CDNA content (Fig. 7C). These results demonstrate that themutation of reas1-ref affects cell proliferation. The first leafof 7-DAG reas1-ref and wild-type seedlings was analyzedby scanning electronmicroscopy to observe the cell size oflower epidermis (Fig. 7D). Therewas significantly smallercell size in reas1-ref than in wild-type seedlings, with cellwidth and cell length both decreased (Fig. 7E). These re-sults demonstrate that cell growth and cell proliferationare suppressed as a result of impaired 60S ribosomematuration, resulting in a developmental delay.

DISCUSSION

reas1-ref/dek* Is a Weak Mutant Allele That FunctionallySuppresses ZmReas1 and Affects 60S Ribosome Biogenesis

Rea1 is a highly conserved ribosome biogenesis fac-tor first identified in the Nug1-purified pre-60S subunit

in yeast, which is the preribosomal particle at the exportfrom the nucleolus to the nucleoplasm (Bassler et al.,2001). The six ATPase modules of Rea1 create a ringdomain, while the large linker and the MIDAS domaincompose the tail structure (Ulbrich et al., 2009). Rea1attaches the preribosome at the Rix1 complex (Rix1-Ipi3-Ipi1) via the ATPase ring domain (Nissan et al.,2002, 2004; Galani et al., 2004). The tail of Rea1 con-taining the MIDAS domain contacts the preribosome atother positions, where the pre-60S factor, Rsa4 or Ytm1,is located. Ytm1 associates with nucleolar pre-60S par-ticles, while Rsa4 associates with nucleoplasmic parti-cles (Bassler et al., 2001; de la Cruz et al., 2005; Mileset al., 2005; Ulbrich et al., 2009). The MIDAS domainof Rea1 interacts with the MIDAS-MIDAS-interactingdomain of Ytm1 or Rsa4; this interaction is essential for60S unit maturation and export from the nucleolus tothe nucleoplasm or from the nucleoplasm to the cyto-plasm, respectively (Bassler et al., 2001; Ulbrich et al.,2009). Rea1 is bound to the pre-60S ribosome at twodistinct sites: one is mediated via the motor ring do-main and the other viaMIDAS interactionwith Ytm1 orRsa4, creating a mechanochemical device to releaseYtm1 or Rsa4 for 60S ribosome export (Kressler et al.,2012).

Compared with dek1, which creates severe effects onkernel development (Becraft et al., 2002), dek* causesonly mild effects. The mutant kernels have an obvioussmall phenotype and decreased seed weight (Fig. 1),with a delay of embryo, endosperm, and seedling de-velopment (Fig. 1). reas1-ref/dek* is a weak, nonlethalmutant allele, where the 2,359th Ala (GCC) of ZmReas1is replaced by Val (GTC; Fig. 2). The reas1mutant allelederived from a Mutator transposon insertion in thecoding region has a defective phenotype and is lethal.reas1-ref is defective at the highly conserved linkageregion and might suppress the function of ZmReas1 byaffecting the mechanical force of the ATPase ring do-main to the MIDAS tail that releases Rsa4 or Ytm1factors. The expression of Reas1 is increased at thetranscript level in reas1-ref, which might be a responseto its functional suppression (Fig. 2).

Table I. (Continued from previous page.)

GO Identifier P Gene DescriptionPolysome/Total in

the Wild Type

Polysome/Total

in reas1

Fold

Change

GRMZM2G108712 Proliferating cell nuclear antigen 0.8651 0.1292 0.15GRMZM2G304362 Ribonucleoside-diphosphate

reductase0.6543 0.0827 0.13

GO: 0033279, ribosomalsubunits

1.16E-03 GRMZM2G099352 40S ribosomal protein S3 0.3527 0.1547 0.44

GRMZM2G078985 40S ribosomal protein S5 0.3491 0.1649 0.47GRMZM2G064640 40S ribosomal protein S9 0.3811 0.1484 0.39GRMZM2G170336 40S ribosomal protein S20 0.4542 0.1796 0.39GRMZM2G110952 40S ribosomal protein S23 0.2834 0.1151 0.41GRMZM2G140609 40S ribosomal protein S23 0.4064 0.1659 0.41GRMZM2G163561 40S ribosomal protein S23 0.4259 0.1402 0.33GRMZM5G868433 60S ribosomal protein L7-2 0.5257 0.2523 0.48GRMZM2G119169 60S ribosomal protein L17 0.3371 0.1421 0.42GRMZM2G091921 60S ribosomal protein L32 0.5604 0.2650 0.48





To our knowledge, the characterization of dek* pro-vides the first description of Rea1 in maize. A significantreduction of mature 60S subunits was observed in yeastrea1 mutants at restrictive conditions (Garbarino andGibbons, 2002; Galani et al., 2004). The Rea1 homologousgene in Arabidopsis is essential for female gametophytedevelopment (Chantha et al., 2010). Rea1AAA-ATPase isconserved from yeast to humans (Bassler et al., 2010).Phylogenetic analysis of ZmReas1 suggests that the Rea1protein is highly conserved in plants and is homologousto proteins in yeast, mammals, andmicroorganisms (Fig.3). A significant reduction of mature 60S ribosomal

subunits is observed in the reas1-ref mutant, consistentwith the effects observed in yeast rea1, indicating a bio-genesis defect specific to the 60S subunit maturationpathway; the maturation and export of 40S subunits arenot affected (Fig. 4). This indicates a conserved functionin 60S subunit biogenesis of Rea1 in yeast and plants.There are reductions of mature 60S subunits in cytoplasmand pre-60S subunits in nucleus (Fig. 4), indicating thatthe nucleus-detained pre-60S subunits might be de-graded, for premature ribosomal particles with biogen-esis failure would be dispersed and degraded in thenucleoplasm (Andersen et al., 2005; Lam et al., 2007).

Figure 7. Evidence of inhibited cell proliferation and cell growth in reas1-ref kernels. A and B, Cell proliferation analysis of 15-and 18-DAPendosperms of the wild type (WT) and reas1-ref. The inset shows the cell cycle diagrams analyzed by flow cytometry.3C and 6C are DNA contents of the nuclei at G1 and S phase of 15- and 18-DAPendosperms, and 12C and 24C are DNA contentsof endoreduplicated nuclei at S phase of 15- and 18-DAP endosperms. For each sample, three independent biological replicateswere performed. Values are means with SE; n = 3 individuals (**, P , 0.01; ***, P , 0.001, Student’s t test). C, Cell proliferationanalysis of 7-DAG seedlings of the wild type and reas1-ref. The inset shows the cell cycle diagrams analyzed by flow cytometry.2C and 4C are DNA contents of the nuclei at G1/S phase and G2/M phase of 7-DAG seedlings. For each sample, three inde-pendent biological replicates were performed. Values are means with SE; n = 3 individuals (**, P , 0.01, Student’s t test). D,Scanning electron microscopy analysis of the lower epidermis of the first leaf mature region of 7-DAG reas1-ref and wild-typeseedlings. S, Stoma. Bars = 50 mm. E, Comparison of cell width and cell length of lower epidermis in wild-type and reas1-ref7-DAG seedlings. The measurements were done on scanning electron microscopy results. Values are means with SE; n = 50individuals (***, P , 0.001, Student’s t test).


Qi et al.



Impaired Ribosome Biogenesis Enhances RibosomeUse Efficiency

The regulation of protein synthesis is a key controlpoint in cellular responses to distinct stresses (Fayeet al., 2014). The proportion of actively translating ri-bosomes is reflected by the percentage of polysomecomplexes/total ribosomes (Branco-Price et al., 2005;Faye et al., 2014). We observed a 1.4-fold increase ofpolysome complexes/total ribosomes in reas1-ref (Fig. 4).This increase in polysomes in reas1-ref is indicative of apromotion in the initiation of protein synthesis. Theproportion of actively translating ribosomes might be inresponse to the down-regulation of ribosome biogenesis.The results of our study of reas1-ref suggest that

suppressed ribosome export enhances ribosome useefficiency and cellular translational efficiency. mRNA,the 40S ribosomal subunit, and eIF2a constitute the 43Spreinitiation complex, termed half-mer, before attach-ment of the 60S ribosomal subunit (Helser et al., 1981;Moy et al., 2002). Multiple eukaryotic protein kinases,each of which responds to different signals, are knownto phosphorylate eIF2a and down-regulate generaltranslation initiation (Chen and London, 1995; Hardinget al., 1999, 2000; Williams, 1999; van Huizen et al.,2003). GCN2 is the only eIF2a kinase found in all eu-karyotes, including plants like Arabidopsis (Berlangaet al., 1999; Zhang et al., 2008). Phosphorylation ofeIF2a is significantly reduced in reas1-ref (Fig. 6), indi-cating increased formation of preinitiation complexesfor protein synthesis, consistent with an increasedproportion of actively translating ribosomes. Further-more, the level of eEF1a protein is markedly increasedin reas1-ref (Fig. 6), suggesting a promotion of both theinitiation and elongation of translation in reas1-ref.Consequently, there is evidence for increased efficiencyof ribosome usage during translation in reas1-ref to en-sure normal rates of protein synthesis (Fig. 8).

Impaired Ribosome Biogenesis Triggers DistinctTranscriptional and Translational Cellular Responses

We have found that the suppressed ribosome matu-ration associated with reas1-ref brings about globalchanges in transcription and translation. The transla-tion of individual mRNAs is regulated, producingdiscrepancies between mRNA and protein levels.mRNAs have a distinct pattern of ribosome loadingunder certain conditions, resulting in altered transla-tional efficiencies (Branco-Price et al., 2005; Gawronet al., 2014). Thus, analysis of transcript level is insuf-ficient to completely describe cellular responses underdifferent conditions. There is also alternative translationthat contributes to the complexity of proteomes (Preisset al., 2003; Serikawa et al., 2003; Blais et al., 2004;Kawaguchi et al., 2004; MacKay et al., 2004; Branco-Price et al., 2005; Lin et al., 2014). According to ourtranscriptome and translatome analysis, there isevidence for consistent and inconsistent transcrip-tional and translational regulation of genes in reas1-ref

endosperm (Fig. 8). The large amount of transcriptionallyup-regulated genes is not the consequence of develop-mental delay, according to the expression data fordeveloping maize kernels (Chen et al., 2014). Ourtranscriptome analysis revealed immediate cellular re-sponses, while the translatome revealed specific proteinchanges that are independent of transcriptional regula-tion for the efficient use of limited ribosomes and energy.

For nucleus-located proteins, the transcription ofsmall and large ribosomal subunit proteins is increasedin reas1-ref endosperm (Fig. 5), whereas their translationis down-regulated (Table I). The transcriptional up-regulation of ribosomal proteins might be a responseto a decreased level of mature ribosomes in the cyto-plasm in reas1-ref (Fig. 4). But increased transcription isincapable of rescuing 60S ribosome export in reas1-ref.Consequently, the percentage of polysome-bound RNAof these genes might be reduced for the more efficientusage of limited ribosomes. Similar to ribosomal pro-teins, there is also a discrepancy in the expression ofnucleosome assembly-related genes. The transcriptionof histones is increased in reas1-ref (Fig. 5), whiletheir translation is down-regulated (Table I). DNAreplication-related genes also have reduced translationin reas1-ref (Table I). The polysome-bound RNA ofhistone- and DNA replication- related genes is mark-edly decreased (Fig. 6), and the histone protein contentis down-regulated in reas1-ref (Fig. 4). The up-regulationof histone transcription might be a response to decreasedgrowth vigor of reas1-ref (Fig. 1) in order to accelerategrowth rate. But accelerated growth might be lethal dueto ribosome shortage. Different kinds of transcriptionalfactors have markedly higher translation in reas1-ref(Table I), including auxin signaling-related genes(GRMZM2G081930 and GRMZM5G809195; Zhanget al., 2014). Among cytosol-located proteins, the tran-scription and translation of translation elongation-related genes are both up-regulated in reas1-ref (Fig. 5;Table I). The final polysome-bound RNA content ofeEF1a and eEF-Tu, as well as the protein level of eEF1a,are markedly increased in reas1-ref (Fig. 6). There arealso inconsistent transcriptional and translational levelsof Reas1 itself (Fig. 2), and it might be a developmentalstage-dependent translational regulation.

In maize kernel, the most abundant protein is zeinstorage protein, which accounts for 50% to 70% of thetotal protein (Holding and Larkins, 2009), and a-zein isthe largest class of zein protein (Heidecker and Mess-ing, 1986). The transcriptionally down-regulated zeinsmight be the consequence of developmental delay.a-Zeins have an increased translation level (Table I);especially the 22-kD a-zeins show markedly increasedprotein in both SDS-PAGE and immunoblot analyses(Figs. 1 and 6). The percentage of a-zein polysome-bound RNA might be increased to ensure basic pro-tein storage in reas1-ref.

The mechanisms that underlie variation in thetranslation of individual genes are likely to involvefeatures of the mRNA sequence (Bailey-Serres andDawe, 1996). Evaluation of highly translated genes





under hypoxia in Arabidopsis showed mRNA se-quences with a low GC nucleotide content in the 59untranslated region (UTR; Branco-Price et al., 2005).RNA 59 UTR GC content, 59 UTR length, and openreading frame length were also observed to affect ri-bosome loading (Jiao and Meyerowitz, 2010; Yángüezet al., 2013). When we analyzed sequence features thatmight affect the ribosome loading of individual genetranscripts affected by the shortage of 60S ribosomesubunits (Supplemental Table S3), no significantly dif-ferent features were observed between the 1,802 up-regulated DTRs and 2,959 down-regulated DTRs,compared with 3,000 randomly selected control genes.However, there might be other feedback pathwaysfor independent regulation at the transcriptional andtranslational levels.

Impaired Ribosome Biogenesis Affects Cell Growthand Proliferation

Cell growth and proliferation are tightly linked, asenhanced protein synthesis is required for cell prolif-eration (Thomas, 2000). The increase in protein syn-thesis is accomplished by an enhanced rate of ribosomebiogenesis in support of the metabolic effort forcell proliferation (Sollner-Webb and Tower, 1986).Normal mitosis includes four successive phases: G1(postmitotic interphase), S (DNA synthesis phase), G2(postsynthetic phase), and M (mitosis; Fowler et al.,1998; Riou-Khamlichi et al., 2000), whereas endoredu-plication of endosperm includes only G1 and S phases(Sabelli and Larkins, 2009). reas1-ref exhibits slowergrowth and cell proliferation (Fig. 7), indicating an

intrinsic link between ribosome biogenesis and cellcycle transition. Based on our analysis, this linkage isthrough the regulation of DNA replication and nucle-osome assembly.

In mammalian cells, the tumor suppressor p53 hasbeen shown to arrest the cell cycle at the G1-S transitionin response to impaired ribosome biogenesis, whilep53-independent cell cycle arrest in response to altera-tion of ribosome biogenesis has also been described(Mayer and Grummt, 2005; Grimm et al., 2006; Zhangand Lu, 2009; Deisenroth and Zhang, 2010; Donati et al.,2011). p53 stabilization leads to cell cycle arrest throughthe regulation of cyclins and cyclin-dependent kinases(Sherr and McCormick, 2002). But the expression levelof cyclins does not appear to be affected in reas1-refaccording to our transcriptome and translatome anal-ysis. The target of rapamycin (TOR) kinase is evolu-tionarily conserved among plant, yeast, and animalcells and is reported to integrate nutrient and energysignaling partly through the phosphorylation of RPS6to promote ribosome biogenesis, polysome accumula-tion, translation initiation, cell growth, and cell prolif-eration (Xiong and Sheen, 2014). The transcription andtranslation levels of TOR signaling-related genes do notappear to be affected in reas1-ref, but there might be analtered phosphorylation level of RPS6 or some otherposttranslational level regulation that is responsible forthe immediate transcriptional up-regulation of genes inreas1-ref. The underlying mechanisms merit furtherexplorations.

S phase is the period for DNA replication, histonesynthesis, and nucleosome assembly. Nucleosome as-sembly is essential for a variety of biological processes,

Figure 8. Suppressed 60S ribosomebiogenesis promotes translation in-itiation and ribosome usage as wellas inconsistently regulates the tran-scription and translation of individualgenes that affect general translationefficiency and cell proliferation. WT,Wild type.


Qi et al.




such as cell cycle progression, development, and senes-cence (Gal et al., 2015). The synthesis of nucleosomeassembly-related proteins (histones and DNA replication-related enzymes) might be reduced to decelerate thegrowth rate for survival under the suppressed ribo-some biogenesis condition in reas1-ref (Table I; Fig. 6).As a result, the nucleosome assembly process duringS phase would be dramatically suppressed. The cellproliferation is slowed in reas1-ref (Fig. 7); thus, togetherwith impaired cell growth, kernel and seedling devel-opment are slowed for more than 3 d (Fig. 1).reas1-ref/dek*, as a nonlethal maize small kernel mu-

tant, offered an opportunity to explore comprehensivecellular responses to impaired 60S ribosome biogenesis.Based on our results, we propose that reduced 60S ri-bosome biogenesis leads to differentially regulatedtranscription and translation of distinct groups of genesthat affect translation efficiency and cell proliferation(Fig. 8). First, there is increased efficiency of translationinitiation and ribosome usage. Second, there is selectivetranslational regulation of different groups of genes forintensive usage of quantitatively limited mature ribo-some. Finally, there is inhibited cell proliferation,leading to slower growth and survival.

MATERIALS AND METHODS

Plant Materials

The o*-N1117mutant stock ofmaize (Zeamays) was obtained from theMaizeGenetics Cooperation stock center and identified as an ethyl methanesulfonate-induced allele of the opaque15 mutant. The dek* mutation was separated fromthe o*-N1117 stock as an independent mutant. The dek* mutant was crossed tothe W64A inbred line, and F1 and F2 were produced to generate a mappingpopulation. All plants were cultivated in the field at the campus of ShanghaiUniversity. Seeds were harvested at 5, 9, 13, 15, 17, 18, 21, 25, and 36 DAP.

Paraffin and Resin Sections

The 15- and 18-DAP embryos were fixed at 4°C overnight in 50% (v/v)ethanol, 5% (v/v) acetic acid, and 3.7% (v/v) formaldehyde (FAA). After em-bedding in paraffin, 10-mmmicrotome sections on glass slides were dewaxed inxylene, rehydrated, and stained with fuchsin. The 15- and 18-DAP endospermtissues were fixed at 4°C overnight in FAA. After embedding in Spurr’s epoxyresin, thin sections (1 mm) were heat fixed to glass slides and stained withfuchsin. Stained sections were rinsed in water three times and air dried. Bright-field photographs of the sections were taken using a Leica microscope.

Transmission Electron Microscopy

The 15- and 18-DAP kernels of reas1-ref and the wild type were preparedaccording to Lending and Larkins (1992), with some modifications: kernelswere fixed in paraformaldehyde and postfixed in osmium tetraoxide. Afterdehydration in an ethanol gradient, samples were transferred to a propyleneoxide solution and gradually embedded in acrylic resin (London Resin). Sec-tions (70 nm)weremadewith a diamond knifemicrotome (Reichert Ultracut E).Sample sections were stained with uranyl acetate, poststained with lead citrate,and observed with a Hitachi H7600 transmission electron microscope.

Scanning Electron Microscopy

The first leaf mature region of 7-DAG reas1-ref and wild-type seedlings wasfixed at 4°C overnight in FAA. Samples were critically dried and spray coatedwith gold. Gold-coated samples were then observed with a scanning electronmicroscope (S3400N; Hitachi).

Protein Quantification

Mature kernels of reas1-ref and the wild type were soaked in water, andendosperm was separated from the embryo and pericarp. Endosperm sampleswere critically dried to constant weight, powdered in liquid N2, and measuredaccording to Wang et al. (2011). Proteins were extracted from 50 mg of threepooled endosperm flour samples. Extracted proteins were measured using abicinchoninic acid protein assay kit (Pierce) according to instructions. Mea-surements of all samples were replicated three times.

Measurement of Starch

Mature kernels of reas1-ref and the wild type were soaked in water, andendosperm was then separated from the embryo and pericarp. Endospermsamples were dried to constant weight and pulverized in liquid N2, and starchwas extracted and measured using an amyloglucosidase/a-amylase methodstarch assay kit (Megazyme) according to the instructions with some modifi-cations: add 0.2 mL of aqueous 80% (v/v) ethanol to a 100-mg sample and aiddispersion; immediately add 3 mL of thermostable a-amylase. Then incubatethe tube in a boiling water bath for 6 min; place the tube in a bath at 50°C; add0.1mL of amyloglucosidase; stir the tube on a vortexmixer and incubate at 50°Cfor 30 min; transfer duplicate aliquots (0.1 mL) of the diluted solution to thebottom; add 3mL of GOPOD reagent and incubate the tubes at 50°C for 20 min;and read the absorbance for each sample and the D-Glc control at 510 nm againstthe reagent blank. The percentage of starch content in mg of dried sample wasanalyzed.

Soluble Amino Acid Analysis

Soluble amino acids were analyzed according to Holding et al. (2010): 3-mgsampleswere refluxed for 24 h in 6 NHCl. Sampleswere hydrolyzed at 110°C for24 h. Sample hydrolysates were critically dried and dissolved in 10mL of citratebuffer. The amino acids were analyzed with a Hitachi-L8900 amino acid ana-lyzer at Shanghai Jiaotong University. Wild-type and dek kernel analyses werereplicated three times.

Map-Based Cloning

The Dek* locus was mapped using 864 individuals from an F2 mappingpopulation of the cross between the dek* and the W64A inbred line. For pre-liminary mapping, molecular markers distributed throughout maize chro-mosome 6 were used. SSR155.1M-1, SSR153.7M-2, SSR154.7M-3, InDel438,InDel428, SNP064, and SNP165 (Supplemental Table S4), as additional molec-ular markers for fine-mapping, were developed to localize the Dek* locus to a101.6-kb region.

RNA Extraction and Real-Time PCR Analysis

Total RNA was extracted with TRIzol reagent (Tiangen), and DNA wasremoved by treatment with RNase-free DNase I (Takara). Using ReverTra Acereverse transcriptase (Toyobo), RNAwas reverse transcribed to complementaryDNA (cDNA). Quantitative real-time PCR was performed with SYBR GreenReal-Time PCR Master Mix (Toyobo) using a Mastercycler ep realplex 2(Eppendorf) according to the standard protocol. Specific primers were designed(Supplemental Table S4), and the experiments were performed with two in-dependent RNA sample sets with ubiquitin as the reference gene. From a poolof kernels collected from three individual plants, an RNA sample was extracted,for which three technical replicates were performed. A final volume of 20 mLcontained 1 mL of reverse transcribed cDNA (1–100 ng), 10 mL of 23 SYBRGreen PCR buffer, and 1.8 mL of 10 mM L21 forward and reverse primers foreach sample. Relative quantifiable differences in gene expressionwere analyzedas described previously (Livak and Schmittgen, 2001).

Fractionation of Cytoplasmic and Nuclear Proteins

Pulverized tissuewas hydrated in cold harvest buffer (10mMHEPES, pH 7.9,50 mM NaCl, 0.5 M Suc, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM dithiothreitol[DTT], 10 mM tetratsodium pyrophosphate, 100 mM NaF, 17.5 mM b-glycero-phosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 4 mg mL21 aprotinin,and 2 mg mL21 pepstatin A) incubated on ice for 5 min, and nuclei was pelleted(1,000 rpm for 10 min). After transfer of the supernatant to a new tube for







extracting cytoplasmic proteins, the pellet of nucleic acid was washed andresuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA,0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 4 mg mL21 aprotinin, and 2 mg mL21 pep-statin A) and pelleted again (1,000 rpm for 10 min). Nuclei were washed andresuspended in buffer C (10 mM HEPES, pH 7.9, 500 mM NaCl, 0.1 mM EDTA,0.1 mM EGTA, 0.1%Nonidet P-40, 1 mMDTT, 1 mM PMSF, 4mgmL21 aprotinin,and 2 mg mL21 pepstatin A) and vortexed (4°C for 15 min), pelleted again(14,000 rpm, 4°C for 10 min), and transferred to a new tube for extracting nu-clear proteins.

Polyclonal Antibodies

For anti-Reas1 antibodyproduction, the 12,478- to 16,038-bp cDNAfragmentwas cloned into pGEX-4T-1 (Amersham Biosciences), and glutathioneS-transferase (GST)-tagged fusion protein was purified with the AKTA purifi-cation system (GE Healthcare) using a GSTrap FF column. Protein expressionand purification followed established procedures. Antibodieswere produced inrabbits according to standard protocols of Shanghai ImmunoGen BiologicalTechnology. For 22- and 19-kD a-zein antibody production, regions of lowsimilarity of 22- and 19-kD a-zein were selected according to a previous study(Woo et al., 2001). The cDNAs responsible for selected polypeptides werecloned into pGEX-4T-1 (Amersham Biosciences), and GST-tagged fusion pro-tein was purified with the AKTA purification system (GE Healthcare) using aGSTrap FF column. Protein expression and purification followed establishedprocedures. Antibodies were produced in rabbits according to standard pro-tocols of Shanghai ImmunoGen Biological Technology.

Immunoblot Analysis

Proteins extracted from reas1-ref and wild-type mature kernels were sepa-rated by SDS-PAGE. Separated protein samples were then transferred to poly-vinylidene difluoride membrane (0.45 mm; Millipore). The membrane with aprotein sample attached on it was incubated with primary and secondary an-tibodies. Using the Super Signal West Pico chemiluminescent substrate kit(Pierce), the signal was visualized according to the manufacturer’s instructions.The purified anti-Reas1 antibody was used at 1:500; the 22- and 19-kD a-zeinantibodies were used at 1:5,000; the a-tubulin antibody (Sigma-Aldrich) wasused at 1:5,000; the Bip (at-95) antibody (Santa Cruz Biotechnology), histoneantibody (Cell Signaling), eRPL13 antibody (Agrisera), eIF2a antibody (CellSignaling), and P-eIF2a antibody (Cell Signaling)were used at 1:1,000; and the TBPantibody (Santa Cruz Biotechnology), eRPS14 antibody (Santa Cruz Biotechnol-ogy), and eEF1a antibody (Santa Cruz Biotechnology) were used at 1:500.

Phylogenetic Analysis

Related sequences were identified in the National Center for BiotechnologyInformation nonredundant protein sequences database by performing aBLASTP search with ZmReas1 protein sequences. Amino acid sequences werealignedwith theMUSCLEmethod in theMEGA5.2 software packageusing theirdefault settings for multiple protein alignment. A rooted phylogenetic tree ofRea1 from maize, Brachypodium distachyon, Triticum urata, Oryza sativa, Setariaitalica, Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Glycine max,Dictyostelium discoideum, Monodelphis domestica, Saprelegnia diclina, Mortierellaverticillata, and Saccharomyces cerevisiaewas constructed by the neighbor-joiningmethod using theMEGA5.2 software package. The evolutionary distances werecomputed using Poisson correction analysis.

RNA-seq Analysis

Total RNA (10 mg) was extracted from endosperm of reas1-ref and wild-typekernels harvested at 15 DAP, and three reas1-ref or wild-type biological sampleswere pooled together. The poly(A) selected RNA-seq library was preparedaccording to Illumina standard instructions (TruSeq Stranded RNA LT Guide).Library DNA was checked for concentration and size distribution in an Agi-lent2100 bioanalyzer before sequencing with an Illumina HiSeq 2500 systemaccording to the manufacturer’s instructions (HiSeq 2500 User Guide). Single-end reads were aligned to the maize B73 genome build Zea mays AGPv2.15using TopHat 2.0.6 (Langmead et al., 2009). Data were normalized as fragmentsper kilobase of exon per million fragments mapped, because the sensitivity ofRNA-seq depends on the transcript length. Significant DTGs were identified asthose with a fold change and P value of differential expression above thethreshold (fold change greater than 2 and P , 0.05).

Ribosome Profile and Isolation of Polysomal RNA

For the ribosome profile, approximately 2.5 mL of pulverized tissue (ap-proximately 20 15-DAP kernels) was hydrated in 2 volumes of polysome ex-traction buffer (PEB; 200 mM Tris-HCl, pH 9, 200 mM KCl, 25 mM EGTA, 35 mM

MgCl2, 1% [w/v] Brij-35, 1% [v/v] Triton X-100, 1% [v/v] Tween 20, 1% [v/v]Igepal CA-630, 1% [w/v] deoxycholic acid, 1% [v/v] polyethylene-10-tridecylether, 1 mM PMSF, 0.5 mg mL21 heparin, 5 mM DTT, 50 mg mL21

cycloheximide, and 50 mg mL21 chloramphenicol; Kawaguchi et al., 2004),homogenized, filtered through two layers of sterile Miracloth (Calbiochem),and cleared by centrifugation (16,000g, 4°C for 15 min). Four hundred A260units of the supernatant was layered over a 15% to 45% (w/v) Suc densitygradient, centrifuged (237,000g, 2.5 h at 4°C; Beckman Optima L-100 XP),and the A254 profile was recorded with a chart recorder using a gradientfractionator connected to a UA-5 detector (BIOCOMP) as described previ-ously (Kawaguchi et al., 2003, 2004). Two independent biological replicateswere performed.

For the isolation of polysomalRNA, approximately 5mLof pulverized tissuepowder (approximately 40 15-DAP kernels) was hydrated in 2 volumes of PEB,homogenized, filtered through two layers of sterile Miracloth (Calbiochem),and cleared by centrifugation (16,000g, 4°C for 15 min). The supernatant waslayered over a 1.75 M Suc cushion (400 mM Tris-HCl, pH 9, 200 mM KCl, 30 mM

MgCl2, 1.75 M Suc, 5 mM DTT, 50 mg mL21 chloramphenicol, and 50 mg mL21

cycloheximide) and centrifuged at 170,000g at 4°C for 3 h (modified fromFennoy and Bailey-Serres, 1995). The polysome pellet was washed withsterile water and resuspended in 700 mL of PEB lacking heparin and deter-gents. Total or polysome-bound RNA was precipitated from total superna-tant or the ribosome fraction of the same amount of sample powder by theaddition of 2.5 volumes of 8 M guanidine chloride and 3.5 volumes of 99%(v/v) ethanol and extracted by use of the Qiagen Plant RNeasy mini kitaccording to the manufacturer’s protocol. For the negative control, the sameamount of pulverized tissue powder was hydrated in 2 volumes of PEB with2 mM puromycin.

The polysome-bound/total RNA value for individual genes was deter-mined from the ratio of the signal in the polysome RNA sample divided by thesignal in the total RNA sample. Due to the required use of an equal RNAquantity in each RNA-seq reaction, in spite of the unequal proportion of RNAin the polysome fraction under the two conditions, it was necessary to nor-malize the signal values obtained for polysome RNA. Normalization wasperformed according to Branco-Price et al. (2005). Polysomes accounted for57.2% and 40.2% of the total absorbance in reas1-ref and wild-type kernels,respectively. The percentage of an individual mRNA species in polysomeswas calculated as 2nP/T (normalized polysome-bound/total) 3 100%.

Normalized polysome-bound/total in reas1-ref:

nP�Tðreas1Þ ¼ log2

ðgene in polysome RNA signalÞðgene in total RNA signalÞ þ log20:5717

Normalized polysome-bound/total in the wild type:

nP�TðWTÞ ¼ log2

ðgene in polysome RNA signalÞðgene in total RNA signalÞ þ log20:4017

Flow Cytometry Detection

For extraction of nuclei, endospermand seedling tissueswerefinely choppedwitha sharp razorblade inBeckman lysis buffer. The resulting slurrywasfilteredthrough a 30-mm nylon filter to eliminate cell debris, and the suspension con-taining nuclei was immediately measured using an Accuri C6 flow cytometer(BD Biosciences) equippedwith an argon-ion laser tuned at a wavelength of 488nm. For each sample, at least 15,000 nuclei were collected and analyzed using alogarithmic scale display. Each flow cytometric histogram was saved and an-alyzed using Accuri C6 software 1.0.264.21 (BD Biosciences).

Sequence data from this article can be found in the GenBank/EMBL datalibraries under the following accession numbers: ZmReas1 KP137367; ZmBip1,NM_001112423, GRMZM2G114793; ZmeRPS6, NM_001112164, GRMZM5G851698;ZmeEF1a, NM_001112117, GRMZM2G153541; ZmHistone H4, NM_001138113,GRMZM2G084195; ZmDNA polymerase «-subunit2, NM_001153609,GRMZM2G154267; and ZmMCM6, NM_001111819, GRMZM2G021069. RNA-seqdata are available from the National Center for Biotechnology InformationGene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) under the series entryGSE67103.


Qi et al.


http://www.ncbi.nlm.nih.gov/geo


Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. SDS-PAGE analysis of total, zein, and nonzeinproteins from dek*/reas1-ref and wild-type mature endosperm.

Supplemental Figure S2. Comparison of total starch content and the per-centage of amylose in wild-type and reas1-ref mature endosperm.

Supplemental Figure S3. Phenotypes of reas1-ref and wild-type plants(90 DAG).

Supplemental Figure S4. Ultrastructure of developing endosperms of thewild type and reas1-ref (15 and 18 DAP).

Supplemental Figure S5. qRT-PCR confirmation of DTRs with increasedor decreased translation levels.

Supplemental Table S1. GO classifications of DTGs with functional anno-tations.

Supplemental Table S2. GO classifications of DTRs with functional anno-tations.

Supplemental Table S3. Sequence feature analysis of DTRs.

Supplemental Table S4. Primers used in these experiments.

ACKNOWLEDGMENTS

We thank Dr. Yuanyuan Ruan (Fudan University) for technical support onthe ribosome profile experiment and Dr. Brian A. Larkins (University ofNebraska, Lincoln) for critical reading of the article.

Received November 13, 2015; accepted December 7, 2015; published December8, 2015.

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