Bioengineering Branch Paternal imprinting of dosageeffect defective1 ded1 contributes to seed weight xenia in maize Dawei Dai Janaki S Mudunkothge Mary Galli Si Nian Char Ruth Davenport ID: 1047317
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1. A. Mark SettlesLife Support Studies ASTBioengineering BranchPaternal imprinting of dosage-effect defective1 (ded1) contributes to seed weight xenia in maizeDawei Dai, Janaki S. Mudunkothge, Mary Galli, Si Nian Char, Ruth Davenport, Xiaojin Zhou, Jeffery L. Gustin, Gertraud Spielbauer, Junya Zhang, W Brad Barbazuk, Bing Yang, Andrea Gallavotti, and A. Mark SettlesAdapted from Nat Commun 13, 5366 (2022). https://doi.org/10.1038/s41467-022-33055-9 under http://creativecommons.org/licenses/by/4.0/Image Credit: NASA
2. Flowering plant seeds result from double fertilization of the haploid generationIn animals, meiosis produces gametes directly.Fertilization results in a zygote → embryo → fetus♀♂Gametes(1n)Adult (2n)Fetus (2n)♀♂Spores (1n)Gametophytes (1n)Sporophyte (2n)In plants, meiosis produces haploid spores.Spores grow mitotically to develop gametophytesMicrospore → 3-cell pollenMegaspore → 7(+)-cell megagametophyteDouble fertilization2n zygote → embryo3n endospermEndosperm has some analogy to a placenta. Transfers nutrients from mother to embryo.Endosperm(3n)Embryo(2n)
3. Maize seed phenotypes show significant parent-of-origin effectsBC1 ears from Mo17xW22 hybridHybrid/W22W22/HybridSame kernel genotypes25% Mo17, 75% W22Hybrid/Mo17Mo17/HybridSame kernel genotypes75% Mo17, 25% W22In 1881, Wilhelm Focke proposed that the paternal influence on seed phenotype had similarity to the ancient Greek concept of xenia.
4. Xenia refers to the sacred responsibilities in the guest-host relationshipIn ancient Greece, hosts had a religious obligation to provide hospitality.One of the obligations of the guest is to give a gift after receiving hospitality.Focke’s idea was that pollen gives some form of genetic gift to the seed:good xenia to produces a healthy seed bad xenia causes poor seed developmentToday, plant xenia is often a catch-all term for any type of paternal-effect on seed development.e.g. complementation of a recessive allele in the maternal plant
5. Imprinting: Different chromatin states allows parent-of-origin specific expressionGregor Mendel discovered dominant-recessive alleles in the 1860’s.When Rr is inherited from the male, expression is suppressed leading to mottled phenotype.Jerry Kermicle discovered imprinting in the 1960’s.Bai & Settles (2015) Front. Plant Sci. 5:780. doi: 10.3389/fpls.2014.00780. PMCID: PMC4307191
6. Parental-conflict or Kinship Theory for the origin of imprintingDiploid angiosperms develop seeds with a 3n endosperm and a 2n embryo.Haig and Westoby (1989) developed a hypothesis to explain this imbalance in the context of imprinting:Maternal genes have selective advantage by optimizing resources to all progenyPaternal genes have selective advantage if they increase resources to their specific progenyTheir prediction for this Parent Specific Gene Expression hypothesis was that there are paternal “guest-gift” genes:There should be genes where the paternally-derived alleles are more strongly expressed than maternally-derived allelesThe genes should encode proteins responsible for acquiring resources from the mother.The genes should be expressed in the offspring tissue that acquires resources.Haig & Westoby (1989) American Naturalist 134: 147-155.SEALEASESRBETLembryo
7. Identifying a guest-gift gene: dosage-effect defective1 (ded1)Can we identify mutants where paternally-derived dek alleles have a quantitative difference? +/+_+ + +Normal weightLight weight+/+mut + mutmutmutmut/mutmutVisible dekmut/mut+mut+Normal weight
8. Quantitative phenotypes: High-throughput single-kernel grain analyzerScreened normal kernels from 1,068 independent dek mutants for quantitative differences in weight or composition
9. Seed weight dosage effect screen of 1,068 independent seed mutants found ded1ded-1/+ Normal kernels segregate 1:1:1+/+dek/++/dekCollect Seed WeightsDai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.Ave. W22 = 206.5 mgAve. Normal = 206.1 mgIncreased variance and a step increase in kernel weight from 197-199 mgEnriched for +/+ genotype
10. Zm00001d033265ded1-refGGACTATTGGAGTATATCTTCTTGTAACTGTAGCTGACCGA100 bpStopded1 encodes an R2R3 Myb transcription factorMap-based cloning identified a retrotransposon insertion in exon 3 of the gene.Dai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
11. Zm00001d033265ded1-refGGACTATTGGAGTATATCTTCTTGTAACTGTAGCTGACCGA100 bpStopded1 encodes an R2R3 Myb transcription factorMap-based cloning identified a retrotransposon insertion in exon 3 of the gene.ded1-1 KRSLKAKVAGded1-2 KRSLKATGGVHPQPVPded1-3 KRSLKATPAEEEEQRAAGAGRAVVRPGGVHPQPVPded1-4 KRSLKAKRRLKKKSNAQQVPAGQWSVLGGVHPQPVPR2 R3 AcidicR2R3Acidic50 aasgRNANLSded1-refCRISPR/Cas9 gene editing generated multiple alleles truncating the open reading frame to delete the acidic activation domain of the protein.Dai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
12. Zm00001d033265ded1-refGGACTATTGGAGTATATCTTCTTGTAACTGTAGCTGACCGA100 bpStopded1-1 KRSLKAKVAGded1-2 KRSLKATGGVHPQPVPded1-3 KRSLKATPAEEEEQRAAGAGRAVVRPGGVHPQPVPded1-4 KRSLKAKRRLKKKSNAQQVPAGQWSVLGGVHPQPVPR2 R3 AcidicR2R3Acidic50 aasgRNANLSded1-refded1-4/ded1-4ded1-4/ded1-refded1-ref/ded1-4Normalded1-ref/ded1-refded1 encodes an R2R3 Myb transcription factorCRISPR alleles have a more severe mutant kernel phenotype.Complementation tests yield intermediate mutant kernel weights.Dai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
13. ded1 has a paternal specific effect on seed weightW22 (♀) X (♂) ded1/+ded1/+ (♀) X (♂) W22Reduces kernel weight with pollen transmission.Normal weight with ear transmission.Paternal weight effect observed in multiple alleles and inbred genetic backgrounds.♀\♂DdDDDDDDDdDDDDDDDd♀\♂DDDDDDDDDDddddDddDDai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
14. ded1 expression is a Paternally Expressed Gene (PEG)Mo17W22⅔W22:⅓Mo1712345678910W22 ♀Mo17♂⅔Mo17:⅓W22Mo17 ♀W22 ♂Ded1Ded1 + AluIMo17W22RT-PCR CAPS assay for relative expression of Ded1.⅔ of Ded1 transcript comes from the single paternal allele.Multiple RNA-seq studies identified ded1 as a PEG:10 DAP: 70-75% paternal mRNA14 DAP: 53-69% paternal mRNAZhang et al (2011) PNAS 108:20042-20047.Waters et al (2011) Plant Cell 23:4221-4233.Zhang et al (2014) Genome Res. 24: 167-176.cbaqRT-PCR Ded1n.d.DDD = ♀ WT X ♂ WTddD = ♀ ded1 X ♂ WTDDd = ♀ WT X ♂ ded1 ddd = ♀ ded1 X ♂ ded1Dai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
15. ded1 is responsible for acquiring resources from the motherBoundpromoters1 kbpfl3DAP-seq identified 5,860 genes with DED1 bound to the promoter regionBoundpromoters2762ded1down1022ded1up10502324764258180870DED1-GSTNor CompetitorMut CompetitorShiftFree---+--++-+-+Overlay with normal vs. ded1 DEGs found 438 direct targets.DED1-activated target genesEnriched for genes with peak expression <14 DAPIncludes: transcription factors, auxin synthesis, sugar transporters6810121416182024222628Endosperm (DAP)DED1-repressed target genesEnriched for genes with peak expression >8 DAPIncludes: starch and anthocyanin accumulationrowminrowmaxDai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
16. ded1 is expressed in offspring tissues that acquire resourcesDed1 is expressed only in endosperm with maximal expression ≤6 DAP.Ded1 is expressed in all endosperm cell types, peak in BETL8 DAPBETLESRSEALCZESource data:Zhan et al (2015) Plant Cell. 27: 513-531.MinMax13 DAPDed1 is expressed in EASSource data:Doll et al (2020) Plant Cell. 32: 833-852.ENDEASDai et al. (2022) Nat Commun. 13(1):5366. doi: 10.1038/s41467-022-33055-9. PMCID: PMC9470594.
17. Conclusion: ded1 is a “guest-gift” xenia effect that exemplifies predictions of kinship theory Haig and Westoby (1989) predictionded1 ObservationEvolutionary selection on imprinting should result in PEGs with paternal bias of expression.Paternal ded1 allele is 50-75% of the transcript.~5-fold greater expression from paternal allele.PEGs should encode proteins responsible for acquiring resources from the mother.DED1 activates genes involved in early endosperm uptake of nutrients.DED1 represses genes involved in latter stages of nutrient storage.PEGs should be expressed in the offspring tissue that acquires resources.Ded1 is expressed exclusively in endosperm.Ded1 expression shifts to the EAS tissue that directs nutrients to the developing embryo.
18. AcknowledgementsFundingNSF-PGRPNIFAChina Scholarship CouncilUF: PMCB, Genetics InstituteVasil-Monsanto EndowmentSettles Lab, UFDawei Dai Janaki MudunkothgeJeff Gustin Gertraud SpielbauerJunya ZhangTechnical SupportFang BaiJohn BaierGina BabbChi-Wah TseungCollaborators & Co-authorsPaul Armstrong, USDA-ARSBrad Barbazuk, U. of FloridaRuth DavenportAndrea Gallavotti, Waksman InstituteMary GalliBing Yang, Donald Danforth CenterSi Nian CharXiaojin Zhou, Chinese Acad. of Ag. Sciences