Modular regulation of floral traits by a PRE1 homolog in Mimulus verbenaceus: implications for the role of pleiotropy in floral integration

Abstract

Floral traits often show correlated variation within and among species. For species with fused petals, strong correlations among corolla tube, stamen, and pistil length are particularly prevalent, and these three traits are considered an intra-floral functional module. Pleiotropy has long been implicated in such modular integration of floral traits, but empirical evidence based on actual gene function is scarce. We tested the role of pleiotropy in the expression of intra-floral modularity in the monkeyflower species Mimulus verbenaceus by transgenic manipulation of a homolog of Arabidopsis PRE1. Downregulation of MvPRE1 by RNA interference resulted in simultaneous decreases in the lengths of corolla tube, petal lobe, stamen, and pistil, but little change in calyx and leaf lengths or organ width. Overexpression of MvPRE1 caused increased corolla tube and stamen lengths, with little effect on other floral traits. Our results suggest that genes like MvPRE1 can indeed regulate multiple floral traits in a functional module but meanwhile have little effect on other modules, and that pleiotropic effects of these genes may have played an important role in the evolution of floral integration and intra-floral modularity.

References

[1] Berg RL . The ecological significance of correlation pleiades. Evolution. 1960; 14: 171-80.

[2] Conner JK . Genetic mechanisms of floral trait correlations in a natural population. Nature. 2002; 420: 407-10.

[3] Edwards CE, Weinig C . The quantitative-genetic and QTL architecture of trait integration and modularity in Brassica rapa across simulated seasonal settings. Heredity. 2011; 106: 661-77.

[4] Fornoni J, Boege K, Pérez-Ishiwara R et al. A comparison of floral integration between selfing and outcrossing species: a meta-analysis. Ann Bot. 2016; 117: 299-306.

[5] Juenger T, Perez-Perez JM, Bernal S et al. Quantitative trait loci mapping of floral and leaf morphology traits in Arabidopsis thaliana: evidence for modular genetic architecture. Evol Dev. 2005; 7: 259-71.

[6] Rose JP, Sytsma KJ . Complex interactions underlie the correlated evolution of floral traits and their association with pollinators in a clade with diverse pollination systems. Evolution. 2021; 75: 1431-49.

[7] Armbruster WS, Pelabon C, Bolstad GH et al. Integrated phenotypes: understanding trait covariation in plants and animals. Philos Trans R Soc B: Biol Sci. 2014; 369: 20130245.

[8] Diggle PK . Modularity and intra-floral integration in metameric organisms: plants are more than the sum of their parts. Phil Trans R Soc B. 2014; 369: 20130253.

[9] Smith SD . Pleiotropy and the evolution of floral integration. New Phytol. 2016; 209: 80-5.

[10] Bissell EK, Diggle PK . Modular genetic architecture of floral morphology in Nicotiana: quantitative genetic and comparative phenotypic approaches to floral integration. J Evol Biol. 2010; 23: 1744-58.

[11] Fishman L, Kelly AJ, Willis JH . Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus . Evolution. 2002; 56: 2138-55.

[12] Feng C, Feng C, Yang L et al. Genetic architecture of quantitative flower and leaf traits in a pair of sympatric sister species of Primulina . Heredity. 2019; 122: 864-76.

[13] Heywood JS, Michalski JS, McCann BK et al. Genetic and environmental integration of the hawkmoth pollination syndrome in Ruellia humilis (Acanthaceae) . Ann Bot. 2017; 119: 1143-55.

[14] Klinkhamer PGL, Van Der Veen-van Wijk CAM . Genetic variation in floral traits of Echium vulgare . Oikos. 1999; 85: 515-22.

[15] Kostyun JL, Gibson MJS, King CM et al. A simple genetic architecture and low constraint allow rapid floral evolution in a diverse and recently radiating plant genus. New Phytol. 2019; 223: 1009-22.

[16] Liao IT, Rifkin J, Cao G et al. Modularity and selection of nectar traits in the evolution of the selfing syndrome in Ipomoea lacunosa (Convolvulaceae) . New Phytol. 2022; 233: 1505-19.

[17] Rosas-Guerrero V, Quesada M, Armbruster WS et al. Influence of pollination specialization and breeding system on floral integration and phenotypic variation in ipomoea . Evolution. 2011; 65: 350-64.

[18] Wessinger CA, Hileman LC, Rausher MD . Identification of major quantitative trait loci underlying floral pollination syndrome divergence in Penstemon . Phil Trans R Soc B. 2014; 369: 20130349.

[19] Lande R . The genetic covariance between characters maintained by pleiotropic mutations. Genetics. 1980; 94: 203-15.

[20] Lande R . The genetic correlation between characters maintained by selection, linkage and inbreeding. Genet Res. 1984; 44: 309-20.

[21] Melo D, Porto A, Cheverud JM et al. Modularity: genes, development, and evolution. Annu Rev Ecol Evol Syst. 2016; 47: 463-86.

[22] Wagner GP, Altenberg L . Perspective: complex adaptations and the evolution of evolvability. Evolution. 1996; 50: 967-76.

[23] Wagner GP, Pavlicev M, Cheverud JM . The road to modularity. Nat Rev Genet. 2007; 8: 921-31.

[24] Wagner GP, Zhang J. The pleiotropic structure of the genotype-phenotype map: the evolvability of complex organisms. Nat Rev Genet. 2011;12:204-13.

[25] Yuan Y-W . Monkeyflowers (Mimulus): new model for plant developmental genetics and evo-devo . New Phytol. 2019; 222: 694-700.

[26] Stanley LE, Ding B, Sun W et al. A tetratricopeptide repeat protein regulates carotenoid biosynthesis and chromoplast development in monkeyflower (Mimulus) . Plant Cell. 2020; 32: 1536-55.

[27] Abraham MC, Metheetrairut C, Irish VF . Natural variation identifies multiple loci controlling petal shape and size in Arabidopsis thaliana . PLoS One. 2013; 8: e56743.

[28] Hepworth J, Lenhard M . Regulation of plant lateral-organ growth by modulating cell number and size. Curr Opin Plant Biol. 2014; 17: 36-42.

[29] Krizek BA, Anderson JT . Control of flower size. J Exp Bot. 2013; 64: 1427-37.

[30] Lee S, Lee S, Yang KY et al. Overexpression of PRE1 and its homologous genes activates gibberellin-dependent responses in Arabidopsis thaliana . Plant Cell Physiol. 2006; 47: 591-600.

[31] Tanaka A, Nakagawa H, Tomita C et al. BRASSINOSTEROID UPREGULATED1, encoding a helix-loop-helix protein, is a novel gene involved in brassinosteroid signaling and controls bending of the lamina joint in rice . Plant Physiol. 2009; 151: 669-80.

[32] Zhao B, Cao JF, Hu GJ et al. Core cis-element variation confers subgenome-biased expression of a transcription factor that functions in cotton fiber elongation. New Phytol. 2018; 218: 1061-75.

[33] Bai MY, Fan M, Oh E et al. A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis . Plant Cell. 2012; 24: 4917-29.

[34] Wang H, Zhu Y, Fujioka S et al. Regulation of Arabidopsis brassinosteroid signaling by atypical basic helix-loop-helix proteins. Plant Cell. 2009; 21: 3781-91.

[35] Zhang L-Y, Bai M-Y, Wu J et al. Antagonistic HLH/bHLH transcription factors mediate brassinosteroid regulation of cell elongation and plant development in rice and Arabidopsis . Plant Cell. 2009; 21: 3767-80.

[36] Chen KY, Cong B, Wing R et al. Changes in regulation of a transcription factor lead to autogamy in cultivated tomatoes. Science. 2007; 318: 643-5.

[37] Heang D, Sassa H . Antagonistic actions of HLH/bHLH proteins are involved in grain length and weight in rice. PLoS One. 2012; 7: e31325.

[38] Stern DL . Evolution, Development, and the Predictable Genome . Greenwood Village, CO: Roberts and Co. Publishers; 2011.

[39] Gurung V, Yuan YW, Diggle PK . Comparative analysis of corolla tube development across three closely related Mimulus species with different pollination syndromes. Evol Dev. 2021; 23: 244-55.

[40] Conner J, Via S . Patterns of phenotypic and genetic correlations among morphological and life-history traits in wild radish. Evolution. 1993; 47: 704-11.

[41] Armbruster W. Floral integration, modularity, and precision: distinguishing complex adaptations from genetic constraints. In: Pigliucci M, Preston K, eds. Phenotypic Integration: Studying the Ecology and Evolution of Complex Phenotypes.Oxford University Press: New York, 2004, 23-49.

[42] Motten AF, Stone JL . Heritability of stigma position and the effect of stigma-anther separation on outcrossing in a predominantly self-fertilizing weed, Datura stramonium (Solanaceae) . Am J Bot. 2000; 87: 339-47.

[43] Opedal ØH, Bolstad GH, Hansen TF et al. The evolvability of herkogamy: quantifying the evolutionary potential of a composite trait. Evolution. 2017; 71: 1572-86.

[44] Wessinger CA, Hileman LC . Accessibility, constraint, and repetition in adaptive floral evolution. Dev Biol. 2016; 419: 175-83.

[45] Ikeda M, Fujiwara S, Mitsuda N et al. A triantagonistic basic helix-loop-helix system regulates cell elongation in Arabidopsis . Plant Cell. 2012; 24: 4483-97.

[46] Bradshaw HD, Otto KG, Frewen BE et al. Quantitative trait loci affecting differences in floral morphology between two species of monkeyflower (Mimulus) . Genetics. 1998; 149: 367-82.

[47] Goodwillie C, Ritland C, Ritland K . The genetic basis of floral traits associated with mating system evolution in Leptosiphon (Polemoniaceae): an analysis of quantitative trait loci. Evolution. 2006; 60: 491-504.

[48] Nakazato T, Rieseberg LH, Wood TE . The genetic basis of speciation in the Giliopsis lineage of Ipomopsis (Polemoniaceae) . Heredity. 2013; 111: 227-37.

[49] Hermann K, Klahre U, Venail J et al. The genetics of reproductive organ morphology in two petunia species with contrasting pollination syndromes . Planta. 2015; 241: 1241-54.

[50] Rifkin JL, Cao G, Rausher MD . Genetic architecture of divergence: the selfing syndrome in Ipomoea lacunosa . Am J Bot. 2021; 108: 2038-54.

[51] Ding B, Patterson EL, Holalu SV et al. Two myb proteins in a self-organizing activator-inhibitor system produce spotted pigmentation patterns. Curr Biol. 2020; 30: 802-814.e8.

[52] Streisfeld MA, Young WN, Sobel JM . Divergent selection drives genetic differentiation in an R2R3-MYB transcription factor that contributes to incipient speciation in Mimulus aurantiacus . PLoS Genet. 2013; 9: e1003385.

[53] Yuan Y-W, Sagawa JM, Young RC et al. Genetic dissection of a major anthocyanin QTL contributing to pollinator-mediated reproductive isolation between sister species of Mimulus . Genetics. 2013; 194: 255-63.

[54] Yuan Y-W, Sagawa JM, Frost L et al. Transcriptional control of floral anthocyanin pigmentation in monkeyflowers (Mimulus) . New Phytol. 2014; 204: 1013-27.

[55] Yuan Y-W, Rebocho AB, Sagawa JM et al. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species . Proc Natl Acad Sci U S A. 2016; 113: 2448-53.

[56] Zheng X, Om K, Stanton KA et al. The regulatory network for petal anthocyanin pigmentation is shaped by the MYB5a/NEGAN transcription factor in Mimulus . Genetics. 2021; 217: iyaa036.

[57] Fishman L, Beardsley PM, Stathos A et al. The genetic architecture of traits associated with the evolution of self-pollination in Mimulus. New Phytol. 2015; 205: 907-17.

[58] Schwarz-Sommer Z, Davies B, Hudson A . An everlasting pioneer: the story of antirrhinum research. Nat Rev Genet. 2003; 4: 657-66.

[59] Vandenbussche M, Chambrier P, Rodrigues BS et al. Petunia, your next supermodel? Front Plant Sci . 2016; 7: 72.

[60] Kramer EM . Aquilegia: a new model for plant development, ecology, and evolution . Annu Rev Plant Biol. 2009; 60: 261-77.

[61] Dong Y, Liu J, Li PW et al. Evolution of Darwin’s peloric gloxinia (Sinningia speciosa) is caused by a null mutation in a pleiotropic TCP gene . Mol Biol Evol. 2018; 35: 1901-15.

[62] Xiao W, Su S, Higashiyama T et al. A homolog of the ALOG family controls corolla tube differentiation in Torenia fournieri . Development. 2019; 146: dev177410.

[63] Zhang R, Fu X, Zhao C et al. Identification of the key regulatory genes involved in elaborate petal development and specialized character formation in Nigella damascena (Ranunculaceae) . Plant Cell. 2020; 32: 3095-112.

[64] Han Y, Burnette JM III, Wessler SR . TARGeT: a web-based pipeline for retrieving and characterizing gene and transposable element families from genomic sequences. Nucleic Acids Res. 2009; 37: e78.

[65] Larsson A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014; 30: 3276-8.

[66] Swofford D. PAUP^∗ Phylogenetic Analysis Using Parsimony (^∗ and Other Methods) Version 4.0 b10: Sinauer Associates . Sunderland, MA; 2002.

[67] Kumar S, Stecher G, Li M et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-9.

[68] Karimi M, Inze D, Depicker A . Gateway vectors for agrobacterium-mediated plant transformation. Trends Plant Sci. 2002; 7: 193-5.

[69] Earley KW, Haag JR, Pontes O et al. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 2006; 45: 616-29.