A single nucleotide mutation of BnaC05.POLIB creates yellow-white chimeric flower in Brassica napus

Abstract

Flower color is a key trait influencing insect pollination and ornamental value, yet the molecular mechanisms underlying heterozygous flower color remain unclear. In this study, we identified the creation of a yellow-white chimeric flower (cf) mutation in Brassica napus, characterized as the coexistence of yellow and white colors on petals of the same flower. Genetic analysis revealed that chimeric flower formation is controlled by a completely dominant gene. Map-based cloning, transgenic complementation, and CRISPR/Cas9 experiments consistently confirmed that BnaC05G0385300ZS on chromosome C05 is the causal gene of CF, which encodes a plastid DNA polymerase IB (BnaC05.POLIB). A G-to-A mutation in the seventh exon results in a D742N substitution, which disrupts Mg²⁺ binding and impairs polymerase activity. This leads to a reduced plastid genome copy number, decreased chromoplast formation, and aberrant carotenoid accumulation, ultimately resulting in the chimeric phenotype in a dosage-dependent manner. These findings reveal a novel role for BnaC05.POLIB in petal color patterning and provide a strategy for breeding ornamental plants with heterozygous flowers.

Acknowledgements

This study was supported by the Biological Breeding-National Science and Technology Major (2022ZD04008) and supported by the Fundamental Research Funds for the Central Universities (2662022ZKYJ004). We thank Dr. H. Chen (Yazhouwan National Laboratory, Sanya, China) and Dr. Y. Song for their critical review of the manuscript and valuable suggestions. We also extend our gratitude to Dr. X. Wang (Department of Andrology/Sichuan Human Sperm Bank, West China Second University Hospital, Sichuan University, Chengdu, China) for his expert guidance on protein structure analysis. The authors declare no conflicts of interest.

Authors contributions

D.H. designed this study. R.X., L.C., P.W., B.H., B.L., and S.L. performed experiments. R.X. performed experimental data analysis and wrote the manuscript. D.H. and G.Y. provided guides about this study. D.H. revised the manuscript.

Data availability

The raw data have been uploaded to the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) under BioProject PRJNA1291401.

Conflicts of interest statement

The authors declare no conflicts of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Cooley AM, Willis JH. Genetic divergence causes parallel evolution of flower color in Chilean Mimulus. New Phytol. 2009; 183:729-39

[2] van der Kooi CJ, Dyer AG, Kevan PG. et al. Functional signifi-cance of the optical properties of flowers for visual signalling. Ann Bot. 2019; 123:263-76

[3] Davies KM, Albert NW, Schwinn KE. From landing lights to mimicry: the molecular regulation of flower colouration and mechanisms for pigmentation patterning. Funct Plant Biol. 2012; 39:619-38

[4] de Jager ML, Ellis AG. Floral polymorphism and the fitness implications of attracting pollinating and florivorous insects. Ann Bot. 2014; 113:213-22

[5] Goodale E, Kim E, Nabors A. et al. The innate responses of bumble bees to flower patterns: separating the nectar guide from the nectary changes bee movements and search time. Naturwissenschaften. 2014; 101:523-6

[6] Simonds V, Plowright CMS. How do bumblebees first find flowers? Unlearned approach responses and habituation. Anim Behav. 2004; 67:379-86

[7] Gronquist M, Bezzerides A, Attygalle A. et al. Attractive and defensive functions of the ultraviolet pigments of a flower (Hypericum calycinum). PNAS. 2001; 98:13745-50

[8] Yuan H, Zhang JX, Nageswaran D. et al. Carotenoid metabolism and regulation in horticultural crops. Hortic Res. 2015; 2:15036

[9] Zhang XP, Xu ZD, Wang WL. et al. Advances on the coloring mechanism of double-color flowers in plants. Hortscience. 2022; 57:1120-7

[10] Li XW, Zheng MM, Gan QQ. et al. The formation and evo-lution of flower coloration in Brassica crops. Front Genet. 2024; 15:1396875

[11] Yilong LI, Taolan Z, Lichao C. et al. Regulation in pigment biosynthesis and color variation of flowers. Chin Bull Life Sci. 2008; 20:147-52

[12] Yan HL, Pei XN, Zhang H. et al. MYB-mediated regulation of anthocyanin biosynthesis. Int J Mol Sci. 2021; 22:3103

[13] Zhao C, Guo W, Chen S. Formation and regulation of flower color in higher plants. Chin Bull Bot. 2005; 22:70-81

[14] Tanaka Y, Sasaki N, Ohmiya A. Biosynthesis of plant pig-ments: anthocyanins, betalains and carotenoids. Plant J. 2008; 54:733-49

[15] Zhang Y, Cheng Y, Xu S. et al. Tree peony variegated flowers show a small insertion in the F3’H gene of the acyanic flower parts. BMC Plant Biol. 2020; 20:211

[16] Lin R-C, Rausher MD.R2R3-MYB genes control petal pig-mentation patterning in Clarkia gracilis ssp. sonomensis (Ona-graceae). New Phytol. 2021; 229:1147-62

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

[18] Todesco M, Bercovich N, Kim A. et al. Genetic basis and dual adaptive role of floral pigmentation in sunflowers. eLife. 2022; 11:11

[19] Yuan Y, Li X, Yao X. et al. Mechanisms underlying the forma-tion of complex color patterns on Nigella orientalis (Ranuncu-laceae) petals. New Phytol. 2023; 237:2450-66

[20] Zhang G, Qi H, Yang S. et al. Research progress of floral organ vacuole pH regulating flower color formation. Biotechnol Bull. 2021; 37:204-10

[21] Morita Y, Hoshino A. Recent advances in flower color variation and patterning of Japanese morning glory and petunia. Breed Sci. 2018; 68:128-38

[22] Martins TR, Berg JJ, Blinka S. et al. Precise spatio-temporal regulation of the anthocyanin biosynthetic pathway leads to petal spot formation in Clarkia gracilis (Onagraceae). New Phytol. 2012; 197:958-69

[23] Gu JW, Guan ZL, Jiao YS. et al. The story of a decade: genomics, functional genomics, and molecular breeding in Brassica napus. Plant Commun. 2024; 5:100884

[24] Fu H, Chao HB, Zhao XJ. et al. Anthocyanins identification and transcriptional regulation of anthocyanin biosynthesis in purple Brassica napus. Plant Mol Biol. 2022; 110:53-68

[25] Cui C, Zhang K, Chai L. et al. Unraveling the mechanism of flower color variation in Brassica napus by integrated metabolome and transcriptome analyses. Front Plant Sci. 2024; 15:1419508

[26] Zhang B, Liu C, Wang Y. et al. Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species. New Phytol. 2015; 206: 1513-26

[27] Liu YJ, Ye SH, Yuan GG. et al. Gene silencing of BnaA09.ZEP and BnaC09.ZEP confers orange color in Brassica napus flowers. Plant J. 2020; 104:932-49

[28] Ye SH, Huang YY, Ma TT. et al. BnaABF3 and BnaMYB44 regulate the transcription of zeaxanthin epoxidase genes in carotenoid and abscisic acid biosynthesis. Plant Physiol. 2024; 195:2372-88

[29] Ye SH, Hua SJ, Ma TT. et al. Genetic and multi-omics anal-yses reveal BnaA07.PAP2In-184-317 as the key gene conferring anthocyanin-based color in Brassica napus flowers. J Exp Bot. 2022; 73:6630-45

[30] Parent J-S. Divergent roles for the two PolI-like organelle DNA polymerases of Arabidopsis. Plant Physiol. 2011; 156: 254-62

[31] Maréchal A, Parent JS, Véronneau-Lafortune F. et al. Whirly proteins maintain plastid genome stability in Arabidopsis. Proc Nat Acad Sci. 2009; 106:14693-8

[32] Udy DB, Belcher S, Williams-Carrier R. et al. Effects of reduced chloroplast gene copy number on chloroplast gene expres-sion in maize. Plant Physiol. 2012; 160:1420-31

[33] Abramson J, Adler J, Dunger J. et al. Accurate structure pre-diction of biomolecular interactions with AlphaFold 3. Nature. 2024; 630:493-500

[34] Doublié S, Tabor S, Long AM. et al. Crystal structure of a bac-teriophage T7 DNA replication complex at 2.2 Å resolution. Nature. 1998; 391:251-8

[35] Parent J-S, Lepage E, Brisson N. Divergent roles for the two polI-like organelle DNA polymerases of Arabidopsis. Plant Physiol. 2011; 156:254-62

[36] Morley SA, Nielsen BL. Chloroplast DNA copy number changes during plant development in organelle DNA polymerase mutants. Front Plant Sci. 2016; 7:7

[37] Zhao C, Safdar LB, Xie M. et al. Mutation of the PHYTOENE DESATURASE 3 gene causes yellowish-white petals in Brassica napus. Crop J. 2021; 9:1124-34

[38] Li FY, Gong YY, Mason AS. et al. Research progress and appli-cations of colorful Brassica crops. Planta. 2023; 258:45

[39] Yu C-Y, Hu S-W, Zhang C-H. et al. The discovery of a novel flower color mutation in male sterile rapeseed (Brassica napus L.). Yichuan. 2004; 26:330-2

[40] Jia LD, Wang JS, Wang R. et al. Comparative transcriptomic and metabolomic analyses of carotenoid biosynthesis reveal the basis of white petal color in Brassica napus. Planta. 2021; 253:8

[41] Li R, Zeng Q, Zhang X. et al. Xanthophyll esterases in associa-tion with fibrillins control the stable storage of carotenoids in yellow flowers of rapeseed (Brassica juncea). New Phytol. 2023; 240:285-301

[42] Sun T, Rao S, Zhou X. et al. Plant carotenoids: recent advances and future perspectives. Mol Hortic. 2022; 2:3

[43] Puthur JT, Shackira AM, Saradhi PP. et al. Chloroembryos: a unique photosynthesis system. J Plant Physiol. 2013; 170:1131-8

[44] Chen H, Zou W, Zhao J. Ribonuclease J is required for chloro-plast and embryo development in Arabidopsis. J Exp Bot. 2015; 66:2079-91

[45] Hsu S-C, Belmonte MF, Harada JJ. et al. Indispensable roles of plastids in Arabidopsis thaliana embryogenesis. Curr Genomics. 2010; 11:338-49

[46] Inaba T, Ito-Inaba Y. Versatile roles of plastids in plant growth and development. Plant Cell Physiol. 2010; 51:1847-53

[47] Lu X, Zhang D, Li S. et al. FtsHi4 is essential for embryogenesis due to its influence on chloroplast development in Arabidop-sis. PLoS One. 2014; 9:e99741

[48] Troesch R, Jarvis P. The stromal processing peptidase of chloroplasts is essential in Arabidopsis, with knockout muta-tions causing embryo arrest after the 16-cell stage. PLoS One. 2011; 6:e23039

[49] Feng J, Fan P, Jiang P. et al. Chloroplast-targeted Hsp90 plays essential roles in plastid development and embryogen-esis in Arabidopsis possibly linking with VIPP1. Physiol Plant. 2014; 150:292-307

[50] Liang Q, Lu X, Jiang L. et al. EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis. Physiol Plant. 2010; 140:380-94

[51] Wang L, Ouyang M, Li Q. et al. The Arabidopsis chloroplast ribosome recycling factor is essential for embryogenesis and chloroplast biogenesis. Plant Mol Biol. 2010; 74:47-59

[52] Bryant N, Lloyd J, Sweeney C. et al. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis. Plant Physiol. 2011; 155:1678-89

[53] Kremnev D, Strand A. Plastid encoded RNA polymerase activ-ity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis. Front Plant Sci. 2014; 5:385-5

[54] Peng J, Harberd NP. Derivative alleles of the Arabidopsis gibberellin-insensitive (gai) mutation confer a wild-type phe-notype. Plant Cell. 1993; 5:351-60

[55] Chen X, Zhang T, Su W. et al. Mutant p53 in cancer: from molecular mechanism to therapeutic modulation. Cell Death Dis. 2022; 13:974

[56] Velten J, Cakir C, Cazzonelli CI. A spontaneous dominant-negative mutation within a 35S::AtMYB90 transgene inhibits flower pigment production in tobacco. PLoS One. 2010; 5:e9917

[57] Duan S, Hu L, Dong B. et al. Signaling from plastid genome sta-bility modulates endoreplication and cell cycle during plant development. Cell Rep. 2020; 32:108019

[58] Steitz TA. DNA polymerases: structural diversity and common mechanisms. J Biol Chem. 1999; 274: 17395-8

[59] Wang PF, Song YX, Xie ZQ. et al. Xiaoyun, a model accession for functional genomics research in Brassica napus. Plant Com-mun. 2024; 5:100727

[60] Song YX, Duan XY, Wang PF. et al. Comprehensive speed breeding: a high-throughput and rapid generation system for long-day crops. Plant Biotechnol J. 2022; 20:13-5

[61] Lichtenthaler HK, Buschmann C Chlorophylls and Carotenoids: measurement and characterization by UV-VIS spectroscopy.Curr. Protoc. Food Anal. Chem. 2001;1:F4.3.1-F4.3.8

[62] Geyer R, Peacock AD, White DC. et al. Atmospheric pressure chemical ionization and atmospheric pressure photoioniza-tion for simultaneous mass spectrometric analysis of micro-bial respiratory ubiquinones and menaquinones. J Mass Spec-trom. 2004; 39:922-9

[63] Yi B, Zeng F, Lei S. et al. Two duplicate CYP704B1-homologous genes BnMs1 and BnMs2 are required for pollen exine for-mation and tapetal development in Brassica napus. Plant J. 2010; 63:925-38

[64] Takagi H, Abe A, Yoshida K. et al. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequenc-ing of DNA from two bulked populations. Plant J. 2013; 74: 174-83

[65] Tang T, Yu XW, Yang H. et al. Development and validation of an effective CRISPR/Cas9 vector for efficiently isolating positive transformants and transgene-free mutants in a wide range of plant species. Front Plant Sci. 2018; 9:1533

[66] Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30:923-30